Methods related to the treatment of neurodegenerative and inflammatory conditions

ABSTRACT

The invention includes methods of neuroprotection, inducing release of neurotrophic factors, inhibiting the over-activation of innate immune cells, attenuating the toxin-induced death and/or damage of tissues, reducing inflammation, treating an inflammation-related condition, and inhibiting NADPH oxidase, that includes contacting or administering an effective amount of at least one compound of the invention that include: valproic acid, sodium butyrate, and salts thereof; opioid peptides; a peptide comprising the tripeptide GGF; and morphinans, such as naloxone, naltrexone, 3-hydroxy-morphinan and dextromethorphan.

This application is application is a National Stage application ofPCT/US2005/016691, filed on 12 May 2005, as a PCT International Patentapplication in the name of The Government of the United States ofAmerica, as represented by the Secretary, Department of Health and HumanServices, applicant for the designation of all countries except theU.S., and Jau-Shyong Hong, Liya Qin, Guorong Li, Michelle Block, WeiZhang, Po-See Chen, and Giia-Sheun Peng, applicants for the designationof the U.S. only, and claims priority to U.S. Provisional App. No.60,570,566, filed May 12, 2004, which is incorporated by referenceherein.

This invention is supported by the Department of Health and HumanServices. The Government of the United States of America may havecertain rights in the invention disclosed and claimed herein below.

FIELD OF THE INVENTION

The invention relates to methods of affecting various biologicalmechanisms related to inflammation, the resultant inflammation and thedisorders that may be caused thereby. More specifically, the inventionrelates to administration of compounds for neuroprotective and/orneurotrophic effects for treatment and/or prevention ofneurodegenerative disorders and diseases caused thereby.

BACKGROUND OF THE INVENTION

Inflammation in the brain is characterized by the activation ofmicroglia and astroglia, and is thought to be associated with thepathogenesis of a number of neurological diseases, including Parkinson'sdisease (PD), Alzheimer's disease and cerebral ischemia. Epidemiologicalstudies have shown a positive correlation between PD and inflammationearly in life. For example, the increase in the incidence of PD in1945-50's was highly correlated to the flu pandemic in 1910-20's. It wasalso found that a higher PD incidence appeared among populations whowere professional boxers at a young age.

It is thought that such primary insults and inflammation activate glialcells, specifically microglia. The activated microglia secrete variouscytokines and free radicals, such as superoxide and nitric oxide (NO),resulting in cerebral inflammation and subsequent neuronal death anddamage. Accumulation and/or overproduction of these factors impactneurons to induce their degeneration.

One specific inflammatory agent that is often implicated in inflammatoryconditions is lipopolysaccharide (LPS). LPS can activate microglia andother cells to overproduce proinflammatory cytokines and free radicals,such as NO, PGE₂, TNFα, superoxide, and other reactive oxygen species(ROS). Cerebral inflammation sustained by microglia activation triggeredby LPS results in a delayed and progressive degeneration of nigradopaminergic neurons. Dopaminergic neurons, in particular, can beespecially vulnerable to oxidative damage due to antioxidant capacity Itis also believed that decreased neurotrophic factor released fromastroglia could play a role in susceptibility to inflammation. Forexample, glial cell line-derived neurotrophic factor (GDNF) issynthesized and released from astroglia. GDNF is believed to at leastpartly mediate neurotrophic effects on dopaminergic (DA) neurons.

Therefore, there remains a need for a greater understanding of themechanisms involved in these disease states and inflammation. There isalso a need for development of agents and treatments that activateneuronal-survival signaling pathways that may enhance the resilience andplasticity of brain cells.

SUMMARY OF THE INVENTION

The invention includes methods of providing neuroprotective and/orneurotrophic effects by contacting, administering, or treating a mammalwith an effective amount of at least one compound of the inventioncomprising valproic acid, sodium valproate, butyric acid, sodiumbutyrate, or other salts thereof; opioid peptides; a peptide comprisingGly-Gly-Phe (GGF); or morphinans, such as naloxone, naltrexone, anddextromethorphan.

An embodiment of the invention provides methods for activatingneuronal-survival signaling pathways in a mammal that compriseadministration of at least one compound of the invention. The inventionprovides methods of inducing release of neurotrophic factors fromastroglial cells by treating the cells with valproic acid, sodiumvalproate, butyric acid, sodium butyrate, or other salts thereof; and/or3-hydroxymorphinan.

The invention also provides methods of inhibiting over activation ofinnate immune cells that comprise contacting an innate immune cell witha therapeutically effective amount of at least one compound of theinvention. The inhibition of the over activation of microglia can occureither in vivo or in vitro.

The invention also includes methods of reducing inflammation in a mammalthat comprise administration of at least one compound of the inventionat an effective dosage.

The invention also includes methods of treating an inflammation-relatedcondition in a mammal that comprises administration of at least onecompound of the invention at an effective dosage to the mammal. Theinflammation-related condition can include inflammation associated withdiseases, such as Alzheimer's disease, Parkinson's disease, ALS,atherosclerosis, diabetes, arthritis, multiple sclerosis, sepsis, septicshock, endotoxemia, multiple organ failure, or organ damage, such asliver damage.

The invention further provides methods for neuroprotection comprisingmethods for reducing inflammation and methods for activatingneuronal-survival signaling pathways.

The invention includes methods of inhibiting the activity of NADPHoxidase that comprise modulating or inhibiting the NADPH oxidase with aneffective amount of at least one compound of the invention. Theinhibition of the activity of NADPH oxidase can occur either in vivo orin vitro.

The invention also includes methods of inhibiting the activity of NADPHoxidase by affecting the gp91 subunit that comprise contacting the gp91subunit with an effective amount of at least one compound of theinvention. The inhibition of the activity of NADPH oxidase can occureither in vivo or in vitro.

The invention also includes methods of inhibiting the activity of NADPHoxidase; inhibiting the NADPH oxidase activity by affecting the gp91subunit; inhibiting the over activation of innate immune cells;decreasing the release of one or more of TNFα; PGE₂, IL-1, nitric oxideor superoxide; attenuating the toxin-induced death and/or damage oftissues; attenuating the toxin-induced deathand/or damage ofdopaminergic neurons; reducing inflammation in a mammal; and treating aninflammation-related condition in a mammal that comprise contacting theparticular enzyme, subunit, cell, tissue, or neuron; or administering tothe mammal an effective amount of a peptide comprising the amino acidsequence GGF (SEQ ID NO:2).

The invention also includes methods of identifying compounds that may betherapeutically effective in treating an inflammation-related conditionthat comprise contacting NADPH oxidase with at least one candidatecompound, and determining whether the candidate inhibits NADPH oxidaseas compared to NADPH oxidase without the compound, wherein a compoundmay be therapeutically effective in treating an inflammation-associatedcondition if the compound decreases the expression or activity of NADPHoxidase or the gp91 subunit of NADPH oxidase.

The invention also includes methods of decreasing the release of one ormore of tumor necrosis factor α (TNFα), prostaglandin E₂ (PGE₂),interleukin-1 (IL-1), nitric oxide, or superoxide that compriseadministration of at least one compound of the invention at an effectivedosage. The decrease in the release of one or more of tumor necrosisfactor α (TNFα), prostaglandin E₂ (PGE₂), interleukin-1 (IL-1), nitricoxide, or superoxide can occur either in vivo or in vitro.

An embodiment of the invention provides a polypeptide or peptidecomprising an amino acid sequence GGF that can be used in methods of theinvention.

The invention also includes compositions that comprise ultra lowconcentrations of a compound of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 is a graph depicting the dopamine (DA) uptake as a percentage ofthe control neuron-glia cells that were pretreated with variousconcentrations of dextromethorphan (DM) followed by lipopolysaccharide(LPS) treatment.

FIG. 2A is a graph representing the number of tyrosine hydroxylase(TH)-immunoreactive neurons in neuron-glia cultures pretreated withvarious concentrations of DM followed by treatment with LPS.

FIG. 2B shows images of immunocytochemically stained dendrite networksthat have and have not been pretreated with various concentrations ofDM.

FIG. 3 is a graph showing DA uptake as a percentage of the control forneuron-glia cells pretreated with various concentrations of DM followedby sequential treatment with Amyloid-β peptide (Aβ).

FIG. 4A is a graph depicting DA uptake as a percentage of the control ofneuron enriched cultures pretreated with various concentrations of DMfollowed by Aβ.

FIG. 4B is a graph depicting DA uptake as a percentage of control ofneuron enriched cultures pretreated with various concentrations of DMfollowed by MPP⁺.

FIG. 5 is a graph depicting photomicrographs of microglia that arepretreated with various concentrations of DM followed by treatment withLPS.

FIGS. 6A, 6B, 6C, 6D, and 6E are graphs depicting the percentage ofLPS-induced increase in the release of nitric oxide (FIG. 6A), PGE₂(FIG. 6B), TNFα (FIG. 6C), superoxide (FIG. 6D), and intracellularreactive oxygen species (iROS) (FIG. 6E) that are pretreated withvarious concentrations of DM.

FIGS. 7A, 7B, and 7C are graphs depicting DA uptake (FIG. 7A), TNF-α(FIG. 7B), and iROS production (FIG. 7C) production in DM pretreated andnon-pretreated PHOX^(+/+) and PHOX^(−/−) mice neuron-glia cultures.

FIGS. 8A and 8B are graphs depicting DA uptake (FIG. 8A), and superoxideproduction (FIG. 8B) in neuron-glia cultures treated with LPS andvarious concentrations of DM post-treatment.

FIG. 9A is an image depicting a Western Blot analysis of iNOS and COX₂production in rat microglia enriched cultures.

FIG. 9B is a graph quantifying the protein levels of FIG. 9A.

FIGS. 10A and 10B are graphs demonstrating nitrite oxide production(FIG. 10A) and PGE₂ production (FIG. 10B) in neuron-glia culturestreated with various concentrations of DM after LPS treatment.

FIG. 11A is a graph illustrating DA uptake of neuron-glia cells with andwithout pretreatment by varying concentrations of the tripeptide GGF andnaloxone followed by treatment with LPS.

FIG. 11B is a graph illustrating the number of TH-IR neurons aftertreatment with varying concentrations of the tripeptide GGF and naloxonefollowed by LPS treatment.

FIG. 11C are photomicrographs of immuno-reactive neurons treated withLPS, LPS plus the tripeptide GGF, and LPS plus naloxone.

FIGS. 12A and 12B are graphs showing the production of iROS (FIG. 12A)and extracellular superoxide (FIG. 12B) of neuron-glia cultures treatedwith GGF or naloxone followed LPS treatment.

FIGS. 13A and 13B are graphs showing DA uptake (FIG. 13A) and TNFαproduction (FIG. 13B) for mesencephalic cultures from PHOX^(+/+) andPHOX^(−/−) mice pretreated with varying concentrations of GGF ornaloxone followed by treatment with LPS.

FIG. 14 is a structural representation comparing of one of the GGFtripeptide conformations (light) superimposed on one of theconformations of naloxone (dark).

FIG. 15 is a graph comparing the binding capacity of naloxone to wildtype cells and cells that do not express the gp91 subunit of NADPH.

FIGS. 16A, 16B, and 16C are photomicrographs of a control mouse liversample (FIG. 16A), a mouse liver sample 12 hours after LPS/galactosamine(GalN) treatment (FIG. 16B), and a mouse treated with DM plus LPS/g GalN(FIG. 16C).

FIG. 17 is a graph showing the level of serum alanine aminotransferase(ALT) in CD-1 mice treated with different amounts of DM.

FIG. 18 is a graph showing the level of serum ALT at different times inCD-1 mice treated with DM.

FIGS. 19A and 19B are graphs depicting serum TNFα (FIG. 19A) and liverTNFα (FIG. 19B) at subsequent times after LPS/GalN injection.

FIGS. 20A and 20B are graphs depicting extracellular superoxide (FIG.20A) and iROS (FIG. 20B) in cells treated with various levels of DM.

FIG. 21 is a graph showing the survival rate of another group of CD-1mice given LPS/GalN and various amounts of DM.

FIG. 22 is a graph showing the levels of serum ALT in the group of CD-1mice from FIG. 21.

FIGS. 23A, 23B, 23C, and 23D are photomicrographs of a mouse liversample treated with LPS/GalN alone (FIG. 23A), a mouse liver sampletreated with 10 mg/kg DM plus LPS/GalN (FIG. 23B), a mouse liver sampletreated with 1 μg/kg DM plus LPS/GalN (FIG. 23C), and a mouse liversample treated with 100 pg/kg DM plus LPS/GalN (FIG. 23D).

FIG. 24 is a graph showing TNFα production in Kupffer cells of CD-1mice.

FIG. 25 depicts DA uptake as a percentage of control of neuron-gliacultures pretreated with various concentrations of valproic acid (VPA)followed by treatment with LPS (*, p<0.05 compared with LPS-treatedcultures; †, p<0.05, ‡, p<0.01, compared with untreated control).

FIG. 26 is a graph illustrating the number of tyrosine hydroxylase(TH)-immunoreactive neurons after treatment with various concentrationsof VPA followed by treatment with LPS (*, p<0.05 compared withLPS-treated cultures; †, p<0.05, ‡, p<0.01, compared with untreatedcontrol).

FIGS. 27A, 27B, 27C, 27D, 27E, and 27F are photomicrographs showingmorphological features of neurons after incubation with vehicle (A);LPS, 20 ng/mL (B); 0.6 mM VPA (C); 0.2 mM VPA, 20 ng/mL LPS (D); 0.4 mMVPA, 20 ng/mL LPS (E); 0.6 mM VPA, 20 ng/mL LPS (F); for 7 days and thenimmunostaining. Scale bar, 25 μm.

FIGS. 28A, 28B, 28C, 28D, 28E, and 28F are photomicrographs showingmorphological features of neuron-enriched cultures treated as indicatedand then immunostained. Scale bar, 100 μm.

FIG. 29 is a graph showing release of TNFα determined 3 hours after LPStreatment.

FIG. 30 is a graph showing levels of nitrite in the supernatant, anindicator of NO production, determined at 24 hours post LPS treatment.

FIG. 31 is a graph showing levels of iROS in enriched microgliadetermined by DCFDA at 2 h after LPS treatment.

FIGS. 32A, 32B, 32C, 32D, 32E, and 32F are photomicrographs showingmorphological features and number of microglia treated with VPA orvehicle for time indicated and then immunostained. Scale bar, 100 μm.

FIG. 33 is a graph showing number of surviving microglia after treatmentwith indicated concentrations of VPA.

FIGS. 34A, 34B, 34C and 34D are photomicrographs showing morphologicalfeatures and number of microglia treated as indicated and thenimmunostained. Scale bar, 100 μm. Scale bar, 100 μm.

FIG. 35 is a graph showing number of surviving microglia after treatmentwith indicated concentrations of VPA.

FIG. 36 is a graph depicting DA uptake as a percentage of control ofneuron-glia cultures pretreated with various concentrations of VPAdose-dependently induces survival-promoting effects against spontaneousDA neuronal death in rat primary mesencephalic neuron-glia cultures.

FIG. 37 is a graph depicting DA uptake as a percentage of control ofneuron-glia cultures pretreated with various concentrations of VPAfollowed by time-dependent treatment with LPS.

FIG. 38 is a graph illustrating the number of tyrosine hydroxylase(TH)-immunoreactive neurons after treatment with various concentrationsof VPA followed by treatment with LPS.

FIG. 39 is a graph showing DA uptake as a percentage of control for ratprimary mesencephalic neuron-glia cultures pretreated with VPA alone,astrocyte conditioned medium (ACM), or ACM-VPA.

FIGS. 40A, 40B, 40C and 40D are photomicrographs showing morphologicalfeatures of neuron-enriched cultures after incubation with vehicle (A),0.6 mM VPA (B), ACM (C) or ACM-VPA (D) for 7 days and thenimmunocytostaining with MAP-2 antibody.

FIGS. 41A and 41B are photomicrographs showing morphological features ofneuron-enriched cultures after incubation with ACM (A) or ACM-VPA (B)for 7 days and then immunocytostaining with TH-IR antibody.

FIG. 42 is a graph showing time-dependent GDNF transcript levelsextracted from rat primary astrocytes and quantitated by real-time PCRrelative to vehicle control following for various times ranging from 6to 48 hours following treatment with VPA.

FIG. 43 is a graph showing secreted GDNF levels from rat primaryastrocytes collected 48 hours after ACM-VPA treatment and analyzed byELISA.

FIG. 44 is a graph showing DA uptake measured seven days after ACM-VPA,pre-incubated overnight with 2 μg/ml of either control goat IgG or goatanti-GDNF IgG, added to mesencephalic neuron-enriched cultures.

FIG. 45 is a graph showing DA uptake in rat primary mesencephalicneuron-glia cultures treated for 48 hours with indicated concentrationsof VPA followed by treatment with 10 ng/ml LPS

FIG. 46 VPA is a graph showing DA uptake in rat primary mesencephalicneuron-glia cultures treated for indicated time with 0.6 mM VPA followedby treatment with 10 ng/ml LPS.

FIGS. 47A, 47B, 47C, 47D, 47E and 47F VPA are images dopaminergicneurons in the primary mesencephalic neuron-glia cultures treated withvehicle alone (A), 0.6 mM VPA alone (B), 10 ng/ml LPS alone (C) orpretreated for 48 hours with 0.2 (D), 0.4 (E) or 0.6 mM VPA (F) followedby treatment with 10 ng/ml LPS and 7 days later immunostained withanti-TH antibody.

FIG. 48 is a graph showing DA uptake in mesencephalic neuron-enrichedcultures pretreated for 48 hours with 0.6 mM VPA followed by treatmentwith 0.5 μM MPP⁺ and [³H]DA uptake was measured 7 days later.

FIG. 49 is a graph showing DA uptake in rat primary mesencephalicneuron-glia cultures seeded in a 24-well culture plate at density of5×10⁵ per well were treated with indicated concentrations of sodiumbutyrate or its vehicle 7 days after seeding.

FIG. 50 is a graph showing DA uptake in midbrain neuron-enrichedcultures 7 days after treatment with vehicle, sodium butyrate, ACM(conditioned medium derived from rat primary astroglial cultures treatedwith vehicle) or ACM-Sodium butyrate (conditioned medium derived fromrat primary astroglial cultures treated with 0.6 mM sodium butyrate) for7 days.

FIG. 51 is a graph showing DA uptake in mesencephalic neuron-gliacultures pretreated for 30 min with 3-hydroxymorphinan (3-HM) (1-5 μM)followed by treatment with 10 ng/mL LPS.

FIG. 52 is a graph showing immunocytochemical analysis, including TH-irneuron counts and neurite length measurements in mesencephalicneuron-glia cultures pretreated for 30 min with 3-HM (1-5 μM) followedby treatment with 10 ng/mL LPS.

FIGS. 53A, 53B, 53C and 53D are representative pictures ofimmunostaining of cells from mesencephalic neuron-glia culturespretreated for 30 min with 3-HM (1-5 μM) followed by treatment with 10ng/mL LPS.

FIG. 54 is a graph showing DA uptake in neuron-enriched cultures treatedwith various concentrations of 3-HM (1-5 μM))

FIG. 55 is a graph showing DA uptake in reconstituted neuron-enrichedcultures with 10% or 20% microglia added and treated with variousconcentrations (1-5 μM) of 3-HM).

FIG. 56 is a graph showing DA uptake in neuron-enriched cultures with40% or 50% astroglia added and treated with various concentrations (1-5μM) of 3-HM.

FIG. 57 is a graph showing DA uptake in neuron-enriched cultures withadded astroglia-derived conditioned media pretreated with variousconcentrations of 3-HM (1-5 μM).

DETAILED DESCRIPTION OF THE INVENTION

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

As used herein, the term “about” applies to all numeric values, whetheror not explicitly indicated. The term “about” generally refers to arange of numbers that one of skill in the art would consider equivalentto the recited value (i.e., having the same function or result). In manyinstances, the term “about” may include numbers that are rounded to thenearest significant figure.

Methods of the invention that comprise steps of contacting a compound ofthe invention with an enzyme or other entity can be accomplished in asolution, or otherwise.

As used herein, “ultra low” amounts or concentrations refers tofemtomolar concentrations (from about 10⁻¹³ M to about 10⁻¹⁵ M) of thecompounds of the invention. Concentrations expressed in M generallycorrespond to a decrease of a dose of about 100 million fold whenadministered in vivo. Effective concentrations for use in methods of theinvention, including ultra low concentrations, are also expressed asgrams per kilogram body weight of the mammal being treated. In general 1mg/kg corresponds to micromolar (1×10⁻⁶ M), and 10 pg/kg generallycorresponds to femtomolar (1×10⁻¹⁵ M concentrations.

As used herein, “pharmaceutically acceptable salt thereof” includes anacid addition salt or a base salt.

As used herein, “pharmaceutically acceptable carrier” includes anymaterial which, when combined with a compound of the invention, allowsthe compound to retain biological activity, such as the ability to treatinflammation associated disease or affect the various mechanismsassociated therewith, and is non-reactive with the subject's immunesystem. Examples include, but are not limited to, any of the standardpharmaceutical carriers such as a phosphate buffered saline solution,water, emulsions such as oil/water emulsions, and various types ofwetting agents. Compositions comprising such carriers are formulated bywell known conventional methods (see, for example, Remington'sPharmaceutical Sciences, Chapter 43, 14th Ed., Mack Publishing Co.,Easton, Pa.).

1. Inhibition of Activity of NADPH Oxidase

The invention includes methods of inhibiting the activity of NADPHoxidase. As used herein, “inhibiting the activity of NADPH oxidase”refers to processes or methods that decrease the activity of NADPHoxidase either in vivo or in vitro, relative to NADPH oxidase either invivo or in vitro, that has not been subjected to such a process ormethod. As used herein “inhibiting the activity of NADPH oxidase”additionally refers to processes or methods that reduce or prevent theover-activity of NADPH oxidase either in vivo or in vitro, relative toNADPH oxidase either in vivo or in vitro, that has not been subjected tosuch a process or method. “Over activity of NADPH oxidase” refers toactivity of this enzyme that is more than that which is commonly seen inan untreated or control subject or cell, whether in vivo or in vitro.

A method of inhibiting the activity of NADPH oxidase comprises a step ofcontacting NADPH oxidase with an effective amount of at least onecompound of the invention. In one embodiment of the invention, the stepof contacting the NADPH oxidase with the at least one compound isaccomplished in vivo. In another embodiment of the invention, the stepof contacting the NADPH oxidase with the at least one compound isaccomplished in vitro.

The invention also includes a method of inhibiting the activity of NADPHoxidase that comprises a step of inhibiting the NADPH oxidase with aneffective amount of a peptide comprising GGF. In some embodiments, apolypeptide or peptide comprises an amino acid sequence GGF(Gly-Gly-Phe) and is other than or excludes the full length sequence ofdynorphin A (SEQ ID NO:1). Preferably, the polypeptide can inhibit theactivity of NADPH oxidase. In other embodiments, a peptide comprisingGGF has no more than 16 amino acids, more preferably, about 3 to 16amino acids, more preferably about 3 to 10 amino acids, and morepreferably about 3 to 5 amino acids. In other embodiments, a peptidethat comprises GGF may be chemically modified or linked to aheterologous polypeptide. In preferred embodiments, a peptide comprisingGGF may be linked to a molecule or compound that enhances intracellulartransport or transport across the blood brain barrier.

The effective amount of the compound is that amount that provides forinhibition of NADPH oxidase activity by at least 25%, more preferably atleast 50%, and most preferably 100%, or to control levels. An inhibitionNADPH oxidase activity can be determined by detecting a decrease inreactive oxygen species (ROS) either extracellularly or intracellularlyor by other methods known to those of skill in the art. In someembodiments, the effect amount can be from about 10⁻⁵ M to about 10⁻¹⁵M. In another embodiment of the invention, the effective amount of thecompound is from about 10⁻⁵ M to about 10⁻⁷ M, or about 10⁻¹³ M to about10⁻¹⁵ M.

In other embodiments, a compound inhibits the activity of NADPH oxidaseif it decreases the activity of NADPH oxidase by at least about 30% whenmeasured by the production of superoxide. In another embodiment of theinvention, a compound inhibits the activity of NADPH oxidase if itdecreases the activity of NADPH oxidase by at least about 50% whenmeasured by the production of superoxide. In another embodiment of theinvention, a compound inhibits the activity of NADPH oxidase if itdecreases the activity of NADPH oxidase by at least about 70% whenmeasured by the production of superoxide.

Over activity of NADPH can be caused by a variety of agents, including,but not limited to lippolysaccharide (LPS), β-amyloid peptides,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), or environmentaltoxins. Examples of environmental toxins include, but are not limited toinsecticides such as rotenone, pesticides, such as paraquat; andparticulate mater (e.g. ubiquitous particulate components of an airpollution).

In other embodiments, other compounds can be utilized to inhibit NADPHoxidase activity. Preferably, the compounds are those that can penetratethe blood brain barrier and act on inflammation in the brain. The othercompounds comprise naloxone, naltrexone, dextromethorphan, valproate,valproic acid or salts thereof, butyric acid or salts thereof, an opioidpeptide such as Met enkephlin or Leu-enkephalin, or mixtures thereof.

In some embodiments, a compound that inhibits NADPH oxidase activity isadministered at an “ultra low” concentration. In some embodiments, theconcentration of the compound is at least 10⁻¹³ M, more preferably about10⁻¹³ M to 10⁻¹⁵ M, more preferably about 10⁻¹⁴ M, and more preferablyabout 10⁻¹⁵ M. In an embodiment, ultra low concentrations comprise about10 pg/kg to about 1000 pg/kg, more preferably about 1000 pg/kg, morepreferably about 100 pg/kg, and more about preferably 10 pg/kg.

In other embodiments, the concentration of the compound in the μM amountis also effective to inhibit NADPH oxidase. In some embodiments, thecompound is administered at about 10⁻⁵ to about 10⁻⁷ M, more preferably10⁻⁵, more preferably 10⁻⁶ and more preferably about 10⁻⁷ M.

In an embodiment, the method comprises contacting NADPH oxidase with aneffective amount of a morphinan or a peptide comprising an amino acidsequence GGF, wherein the effective amount is about 10⁻¹³ to about 10⁻¹⁵M. Morphinans include without limitation, dextromethorphan, naloxone,and naltrexone. A peptide comprising an amino acid sequence GGF,preferably, has no more than sixteen amino acids and does not includethe full length sequence of dynorphin A (SEQ ID NO:1). Examples of somepeptides comprising GGF are shown in Table 1.

NADPH oxidase is a complex enzyme that contains 7 subunits, one of whichis gp91. gp91 is the catalytic subunit of NADPH, and therefore may playa significant role in modulating the activity of NADPH.

The invention therefore also includes methods of inhibiting the activityof NADPH oxidase by affecting the gp91 subunit of NADPH oxidase. As usedherein, “affecting the gp91 subunit of NADPH oxidase” refers toprocesses or methods that alter the configuration of one or more regionsof the gp91 subunit either in vivo or in vitro, relative to a gp91subunit either in vivo or in vitro, that has not been subjected to sucha process or method; or processes or methods that block at least aportion of the gp91 subunit from binding with another protein orcompound either in vivo or in vitro, relative to a gp91 subunit eitherin vivo or in vitro, that has not been subjected to such a process ormethod.

A method of affecting the gp91 subunit of NADPH oxidase comprises thestep of contacting the gp91 subunit of NADPH oxidase with an effectiveamount of at least one compound of the invention. In one embodiment ofthe invention, the step of contacting the gp91 subunit of NADPH oxidasewith the at least one compound is accomplished in vivo. In anotherembodiment of the invention, the step of contacting the gp91 subunit ofNADPH oxidase with the at least one compound is accomplished in vitro.

The invention also includes methods of affecting the gp91 subunit ofNADPH oxidase that comprises the step of contacting the gp91 subunitwith an effective amount of a peptide comprising tripeptide GGF. In someembodiments, a polypeptide or peptide that comprises an amino acidsequence GGF (Gly-Gly-Phe) is other than or excludes the full lengthsequence of dynorphin A (SEQ ID NO:1). Preferably, the polypeptide caninhibit the activity of NADPH oxidase. In other embodiments, a peptidecomprising GGF has no more than 16 amino acids, more preferably, about 3to 16 amino acids, more preferably about 3 to 10 amino acids, and morepreferably about 3 to 5 amino acids. In other embodiments, a peptidethat comprises GGF may be chemically modified or linked to aheterologous polypeptide. In preferred embodiments, a peptide comprisingGF may be linked to a molecule or compound that enhances intracellulartransport or transport across the blood brain barrier. In one embodimentof the invention, the effective amount of the compound can be from about10⁻⁵ M to about 10⁻¹⁵ M. In another embodiment of the invention, theeffective amount of the compound is from about 10⁻⁵ M to about 10⁻⁷ M,or about 10⁻¹³ M to about 10⁻¹⁵ M.

NADPH controls the release of pro-inflammatory agents from the innateimmune cells within a tissue system. Examples of innate immune cellswithin particular tissue systems include, but are not limited tomicroglia in neurological tissues, macrophages in organs, such as forexample Kupffer cells in the liver, macrophages in the lungs, masengialcells in the kidney, and the endothelial cells lining the blood. Theinvention additionally includes methods of inhibiting microglial NADPHoxidase.

It is thought that preventing or reducing the amount of over-activity ofNADPH oxidase can reduce the over-activation of the innate immune cellswithin a particular tissue system. The invention also includes methodsof inhibiting activation of at least one innate immune cell within atissue system. The invention further includes methods of inhibitingmicroglial activation. Another embodiment of the invention providesmethods of inhibiting the activation or over-activation of at least oneinnate immune cell that comprise contacting the innate immune cell withan therapeutically effective amount of at least one compound of theinvention. In one embodiment of the invention, the step of contactingthe innate immune cell with the at least one compound is accomplished invivo. In another embodiment of the invention, the step of contacting theinnate immune cell with the at least one compound is accomplished invitro.

An embodiment of the invention provides methods of inhibiting theactivation or over-activation of at least one innate immune cell thatcomprise contacting the innate immune cell with an effective amount of apeptide or polypeptide comprising tripeptide GGF. In some embodiments, apolypeptide or peptide comprises an amino acid sequence GGF(Gly-Gly-Phe) and is other than or excludes the full length sequence ofdynorphin A (SEQ ID NO:1). Preferably, the polypeptide can inhibit theactivity of NADPH oxidase and/or the production of reactive oxygenspecies. In other embodiments, a peptide comprising GGF has no more than16 amino acids, more preferably, about 3 to 16 amino acids, morepreferably about 3 to 10 amino acids, and more preferably about 3 to 5amino acids. In other embodiments, a peptide that comprises GGF may bechemically modified or linked to a heterologous polypeptide. Inpreferred embodiments, a peptide comprising GGF may be linked to amolecule or compound that enhances intracellular transport or transportacross the blood brain barrier. In one embodiment of the invention, theeffective amount of the compound can be from about 10⁻⁵ M to about 10⁻¹⁵M. In another embodiment of the invention, the effective amount of thecompound is from about 10⁻⁵ M to about 10⁻⁷ M, or ultra lowconcentrations of about 10⁻¹³ M to about 10⁻¹⁵ M. In an embodiment,ultra low concentrations comprise about 10 pg/kg to about 1000 pg/kg,more preferably about 1000 pg/kg, more preferably about 100 pg/kg, andmore about preferably 10 pg/kg.

Yet another embodiment of the invention provides methods of inhibitingthe activation or over-activation of at least one microglial cell thatcomprises contacting the microglial with an effective amount of at leastone compound of the invention. In one embodiment of the invention, thestep of contacting the microglial with the at least one compound isaccomplished in vivo. In another embodiment of the invention, the stepof contacting the microglial with the at least one compound isaccomplished in vitro.

In other embodiments, other compounds can be utilized to inhibitactivation of innate immune cells. Preferably, the compounds are thosethat can penetrate the blood brain barrier and act on inflammation inthe brain. The other compounds comprise naloxone, naltrexone,dextromethorphan, valproate, valproic acid or salts thereof, butyricacid or salts thereof, an opioid peptide such as Met enkephalin orLeu-enkephalin, or mixtures thereof.

An embodiment of the invention provides methods of inhibiting activationof at least one innate immune cell, comprising contacting the cell withan effective amount of a compound of the invention. In an embodiment,the method of inhibiting activation decreases the activity of the atleast one innate immune cell by at least about 30%. In an embodiment,the method of inhibiting activation decreases the activity of the atleast one innate immune cell by at least about 50%. In an embodiment,the method of inhibiting activation decreases the activity of the atleast one innate immune cell by at least about 70%.

The invention also includes methods of inhibiting the activation orover-activation of at least one microglial cell that comprisescontacting the microglial with an effective amount of tripeptide GGF. Inone embodiment of the invention, the effective amount of the compoundcan be from about 10⁻⁵ M to about 10⁻¹⁵ M. In another embodiment of theinvention, the effective amount of the compound is from about 10⁻⁵ M toabout 10⁻⁷ M, or about 10⁻¹³ M to about 10⁻¹⁵ M.

As used herein, the phrase “inhibiting microglial activation” refers toprocesses or methods that deactivate previously activated microgliaeither in vivo or in vitro, relative to microglia either in vivo or invitro, that have not been subjected to such a process or method;processes or methods that slow the activation of microglia either invivo or in vitro, relative to microglia either in vivo or in vitro, thathave not been subjected to such a process or method; processes ormethods that limit the number of microglia that are activated, either invivo or in vitro, relative to microglia either in vivo or in vitro, thathave not been subjected to such a process or method; or processes ormethods that lessen the level of activation of activated microgliaeither in vivo or in vitro, relative to microglia either in vivo or invitro, that have not been subjected to such a process or method.

The activation of innate immune cells, such as microglia or Kupffercells involves the release of a number of soluble pro-inflammatoryfactors, including, but not limited to tumor necrosis factor alpha(TNFα), prostaglandin E₂ (PGE₂), interleukin-1 (IL-1), and free radicalssuch as nitric oxide and superoxide.

A compound may be determined to have an ability to “inhibit NADPHactivity”, “affect the gp91 subunit”, or “inhibit activation of innateimmune cells” by measuring and/or monitoring at least one of thefollowing: tumor necrosis factor alpha (TNFα), prostaglandin E₂(_(PGE2)), interleukin-1 (IL-1), free radicals such as nitric oxide (NO)and superoxide (O₂ ⁻), and the immunostaining intensity of OX-42immunoreactivity, which is a marker for the activation of microglia.

In another embodiment, the invention also includes methods of decreasingthe release of one or more of TNFα, PGE₂, IL-1, NO and O₂ ⁻ from innateimmune cells, such as microglia.

TNFα

One example of a method of measuring and/or monitoring the amount ofTNFα in tissues and or serum includes a TNFα enzyme-linked immunosorbentassay (ELISA) kit. An example of one such kit is TNFα-ELISA kit, whichis commercially available from R&D Systems, Minneapolis Minn. One ofskill in the art, having read this specification would understand andrealize what other methods could be used to monitor the amount of TNFαin tissue, serum, or some combination thereof. The invention alsoenvisions and encompasses use of such other methods to monitor and/ormeasure the amount of TNFα in samples.

PGE₂

One example of a method of measuring and/or monitoring the amount ofPGE₂ in tissues and or serum includes prostaglandin E₂ enzymeimmunoassay (EIA) kit. An example of one such kit is PGE₂-ELISA kit,which is commercially available from Cyaman, Ann Arbor, Mich. One ofskill in the art, having read this specification would understand andrealize what other methods could be used to monitor the amount of PGE₂in tissue, serum, or some combination thereof. The invention alsoenvisions and encompasses use of such other methods to monitor and/ormeasure the amount of PGE₂ in samples.

IL-1

One example of a method of measuring and/or monitoring the amount ofIL-1 in tissues and or serum includes an IL-1 enzyme-linkedimmunosorbent assay (ELISA) kit. An example of one such kit is IL-1ELISA kit, which is commercially available from R&D Systems, MinneapolisMinn. One of skill in the art, having read this specification wouldunderstand and realize what other methods could be used to monitor theamount of IL-1 in tissue, serum, or some combination thereof. Theinvention also envisions and encompasses use of such other methods tomonitor and/or measure the amount of IL-1 in samples.

Nitric Oxide

One example of a method of measuring and/or monitoring the amount ofnitrite (which relates to nitric oxide) in tissues and or serum includesmeasuring the accumulated levels of nitrite in the supernatant with theGriess reagent. (Green et al., 1982) Griess reagent kits are availablecommercially, for example from Promega Corporation, Madison, Wis. One ofskill in the art, having read this specification would understand andrealize what other methods could be used to monitor the amount of nitricoxide and/or nitrite in tissue, serum, or some combination thereof. Theinvention also envisions and encompasses use of such other methods tomonitor and/or measure the amount of nitric oxide and/or nitrite insamples.

Superoxide Production

A compound may be determined to have an ability to “affect the gp91subunit” or “inhibit the activity of NADPH oxidase” by monitoring theamount of superoxide in tissues or serum. One example of a method ofmeasuring and/or monitoring the amount of superoxide (O₂ ⁻) in tissuesand or serum includes a method of measuring the superoxide dismutase(SOD) inhibitable reduction of tetrazolium salt, WST-1.

One example of a specific method to measure the immediate release ofsuperoxide from microglia-enriched or neuron-glia after stimulation, isto grow cultures in for example, a 96-well plate in a 10% maintenancemedium, and switch them to phenol red-free HBSS (50 μl/well). To eachwell 50 μl of HBSS containing the compound whose effect is to bedetermined is added. The cultures can then be incubated at about 37° C.for about 30 min followed by the addition of about 50 μ offerricytochrome c (100 μM) in HBSS, with and without 600 U/ml superoxidedismutase (SOD), 50 μl of vehicle or lipopolysacchride (LPS) in HBSS.The absorbance at 550 nm can then be read with a microplatespectrophotometer, such as a SpectraMax Plus device availablecommercially from Molecular Devices in Sunnyvale, Calif. One of skill inthe art will also understand that other similar methods and variants ofthis method can also be used to measure the superoxide production.

One of skill in the art, having read this specification would understandand realize that other methods could be used to monitor the amount ofsuperoxide in tissue, serum, or some combination thereof. The inventionalso envisions and encompasses use of such other methods to monitorand/or measure the amount of superoxide in samples.

In one embodiment of a method of the invention, the tissues that can bemonitored for the various pro-inflammatory factors mentioned herein (aswell as others that have the same indications, as would be known to oneof skill in the art having read this specification) include, but are notlimited to brain, and liver tissues. In another embodiment of theinvention, serum levels can be monitored for the various componentsmentioned herein (as well as others that have the same indications, aswould be known to one of skill in the art having read thisspecification).

In one embodiment of the invention, a compound inhibits NADPH activity,affects the gp91 subunit, inhibits overactivation or activation ofinnate immune cells such as microglia if it decreases the release of oneor more of TNFα, PGE₂, IL-1, NO, or O₂ ⁻ by at least about 30% whenmeasured using a method known to those of skill in the art having readthis specification. In one embodiment of the invention, a compoundinhibits NADPH activity, affects the gp91 subunit, inhibitsoveractivation or activation of innate immune cells such as microglia ifit decreases the release of one or more of TNFα, PGE2, IL-1, NO₂, or O₂⁻ by at least about 50% when measured using a method known to those ofskill in the art having read this specification. In one embodiment ofthe invention, a compound inhibits NADPH activity, affects the gp91subunit, inhibits overactivation or activation of innate immune cellssuch as microglia if it decreases the release of one or more of TNFα,PGE₂, IL-1, NO, or O₂ ⁻ by at least about 70% when measured using amethod known to those of skill in the art having read thisspecification.

2. Methods of Inhibiting Toxin-Induced Death or Damage of DopaminergicNeurons

The invention also includes methods of attenuating or inhibitingtoxin-induced death and/or damage of cells, such as neurons, livercells, lung cells, and kidney cells. Examples of toxins that can inducedopaminergic neuron death and/or damage include, but are not limited toLPS, Aβ peptides (amyloid peptides), and environmental toxins.

LPS is an endotoxin from the outer membrane of the majority of thegram-negative bacteria, and may have implications in sepsis, organfailure and shock. LPS can induce septic shock in laboratory animals.Kupffer cells, resident macrophages in the liver, remove bacteria andtheir related endotoxins from the body when activated. In turn, theactivated Kupffer cells release active substances, such as freeradicals, and inflammatory cytokines. Examples of such free radicals andinflammatory cytokines include, but are not limited to tumor necrosisfactor alpha (TNFα), prostaglandin E₂ (PGE₂), interleukin-1 (IL-1), andfree radicals such as nitric oxide and superoxide. Reduction of suchfree radicals and inflammatory cytokines may therefore assist indecreasing the likelihood, occurrence, or severity of endotoxemia,septic shock, and multiple organ failure.

As used herein, the phrase attenuating toxin-induced death and/or damageof cells, such as neurons, refers to processes or methods that lessenthe number of cells that die and/or are damaged as a result of one ormore toxins either in vivo or in vitro, relative to cells either in vivoor in vitro, that have not been subjected to such a process or method;or processes or methods that lessen the severity of the effects of oneor more toxins on the cells either in vivo or in vitro, relative tocells either in vivo or in vitro, that have not been subjected to such aprocess or method.

In one embodiment of the invention, a compound attenuates toxin-induceddeath and/or damage of cells if it decreases the percentage of cellsthat die and/or are damaged as a result of the toxin, relative totissues not treated with the compound by a statistically significantamount. In another embodiment of the invention, a compound attenuatestoxin-induced death and/or damage of cells if it decreases thepercentage of cells that die as a result of the toxin, relative to cellsnot treated with the compound by at least about 30%. In yet anotherembodiment of the invention, a compound attenuates toxin-induced deathof cells if it decreases the percentage of cells that die as a result ofthe toxin, relative to cells not treated with the compound by at leastabout 50%. In yet another embodiment of the invention, a compoundattenuates toxin-induced death of cells if it decreases the percentageof cells that die as a result of the toxin, relative to cells nottreated with the compound by at least about 70%.

Methods of attenuating or inhibiting toxin-induced death and/or damageof cells comprise the step of contacting at least one cell with aneffective amount of at least one compound of the invention. In a furtherembodiment, the method comprises contacting at least one immune orinflammatory cell with an effective amount of at least one compound ofthe invention. In one embodiment of the invention, the step ofcontacting the at least one immune cell with the at least one compoundis accomplished in vivo. In another embodiment of the invention, thestep of contacting the at least one immune cell with the at least onecompound is accomplished in vitro.

The invention also includes methods of attenuating toxin-induced deathof innate immune cells that comprise the step of contacting at least oneinnate immune cell with an effective amount of a peptide comprising anamino acid sequence tripeptide GGF. In some embodiments, a polypeptideor peptide comprises an amino acid sequence GGF (Gly-Gly-Phe) and isother than or excludes the full length sequence of dynorphin A (SEQ IDNO:1). Preferably, the polypeptide can inhibit the activity of NADPHoxidase. In other embodiments, a peptide comprising GGF has no more than16 amino acids, more preferably, about 3 to 16 amino acids, morepreferably about 3 to 10 amino acids, and more preferably about 3 to 5amino acids. In other embodiments, a peptide that comprises GGF may bechemically modified or linked to a heterologous polypeptide. Inpreferred embodiments, a peptide comprising GGF may be linked to amolecule or compound that enhances intracellular transport or transportacross the blood brain barrier.

In one embodiment of the invention, the effective amount of the compoundcan be from about 10⁻⁵ M to about 10⁻¹⁵ M. In another embodiment of theinvention, the effective amount of the compound is from about 10⁻⁵ toabout 10⁻⁷, or about 10⁻¹³ to about 10⁻¹⁵ M. In some embodiments, theeffective amount of the compound is at least about 10 pg/kg. Inadditional embodiments, the effective amount of the compound is at leastabout 100 pg/kg to about 10 mg/kg. In a further embodiment, theeffective amount is at least about 100 pg/kg to about 1 μg/kg. Inanother embodiment the effective amount is from about 1 μg/kg to about10 mg/kg. In a still further embodiment, the effective amount is fromabout 5 mg/kg, more preferably about 6 mg/kg to about 25 mg/kg.

In other embodiments, other compounds can be utilized to inhibit orattenuate toxin induced death and/or damage of cells. Reduction ofreactive oxygen species and/or clearance of TNF-α are factors inprotecting cells and tissues from toxin associated damage. Compoundsthat inhibit production and/or activity of these mediators are useful totreat or inhibit toxin induced death or damage of cells. Such compoundscomprise naloxone, naltrexone, dextromethorphan, valproate, valproicacid or salts thereof, butyric acid or salts thereof, an opioid peptidesuch as Met enkephalin or Leu-enkephalin, or mixtures thereof.

3. Reduction of Inflammation

The invention also includes methods of reducing inflammation thatcomprise administering an effective amount of at least one of thecompounds of the invention to a mammal.

The invention also includes methods of reducing inflammation thatcomprise administering to a mammal or human subject in need thereof aneffective amount of a peptide comprising an amino acid sequence GGF. Insome embodiments, a polypeptide or peptide comprises an amino acidsequence GGF (Gly-Gly-Phe) and is other than or excludes the full lengthsequence of dynorphin A (SEQ ID NO:1). Preferably, the polypeptide caninhibit the activity of NADPH oxidase. In other embodiments, a peptidecomprising GGF has no more than 16 amino acids, more preferably, about 3to 16 amino acids, more preferably about 3 to 10 amino acids, and morepreferably about 3 to 5 amino acids. In other embodiments, a peptidethat comprises GGF may be chemically modified or linked to aheterologous polypeptide. In preferred embodiments, a peptide comprisingGGF may be linked to a molecule or compound that enhances intracellulartransport or transport across the blood brain barrier.

In one embodiment of the invention, the effective amount of the compoundcan be from about 10⁻⁵ M to about 10⁻¹⁵ M. In another embodiment of theinvention, the effective amount of the compound is from about 10⁻⁵ M toabout 10⁻⁷ M, or about 10⁻¹³ M to about 10⁻¹⁵ M.

In some embodiments, the method of reducing inflammation comprisesadministering an effective amount at least one morphinan to a mammal orhuman subject in need thereof. Morphinans include, without limitation,dextromethorphan, naloxone, and naltrexone. In further embodiments, theeffective amount is an ultra low concentration. Examples of ultra lowconcentration comprise about 10⁻¹³ to 10⁻¹⁵ M, more preferably 10⁻¹³ M,more preferably 10⁻¹⁴ M, and more preferably 10⁻¹⁵ M. In an embodiment,ultra low concentrations comprise about 10 pg/kg to about 1000 pg/kg,more preferably about 1000 pg/kg, more preferably about 100 pg/kg, andmore about preferably 10 pg/kg.

In some embodiments, the method of reducing inflammation comprisesadministering an effective amount of valproate, valproic acid, butyricacid, sodium valproate, sodium butyrate or other salts thereof, to amammal or human subject in need thereof. In further embodiments, theeffective amount is an ultra low concentration. Examples of ultra lowconcentration comprise about 10⁻¹³ to 10⁻¹⁵ M, more preferably 10⁻¹³ M,more preferably 10⁻¹⁴ M, and more preferably 10⁻¹⁵ M. In an embodiment,ultra low concentrations comprise about 10 pg/kg to about 1000 pg/kg,more preferably about 1000 pg/kg, more preferably about 100 pg/kg, andmore about preferably 10 pg/kg.

In some embodiments, the method of reducing inflammation comprisesadministering to a mammal or human subject in need thereof at least oneopioid peptide. Opioid peptides, include, without limitation, leuenkephalin and/or met enkephalin. In further embodiments, the effectiveamount is an ultra low concentration. Examples of ultra lowconcentration comprise about 10⁻¹³ to 10⁻¹⁵ M, more preferably 10⁻¹³ M,more preferably 10⁻¹⁴ M, and more preferably 10⁻¹⁵ M. In an embodiment,ultra low concentrations comprise about 10 pg/kg to about 1000 pg/kg,more preferably about 1000 pg/kg, more preferably about 100 pg/kg, andmore about preferably 10 pg/kg.

In some embodiments, a method of reducing inflammation comprisesadministering to a mammal or human subject in need thereof a peptidecomprising an amino acid sequence GGF. Peptides comprising GGF,preferably do not include the full length sequence of dynorphin A (SEQID NO:1). Examples of some peptide comprising GGF are shown in Table 1.In some embodiments, peptides comprise no more than 16 amino acids,preferably about 3 to 16 amino acids, more preferably about 3 to 10amino acids, and more preferably about 3-5 amino acids. In someembodiments, a peptide comprising GGF, further comprises anothercompound or a heterologous polypeptide. In further embodiments, theeffective amount is an ultra low concentration. Examples of ultra lowconcentration comprise about 10⁻¹³ to 10⁻¹⁵ M, more preferably 10⁻¹³ M,more preferably 10⁻¹⁴ M, and more preferably 10⁻¹⁵ M. In an embodiment,ultra low concentrations comprise about 10 pg/kg to about 1000 pg/kg,more preferably about 1000 pg/kg, more preferably about 100 pg/kg, andmore about preferably 10 pg/kg.

An inflammatory condition can exist as a result of many factors. In someembodiments, the inflammatory condition is associated or related todisease or disorder including Alzheimer's disease, Parkinson's disease,ALS, MS, atherosclerosis, diabetes, arthritis, sepsis, septic shock,endotoxemia, multiple organ failure or organ damage.

As used herein, the phrase “reduce inflammation” includes lessening atleast one physiological effect of inflammation, lessening at least onesymptom associated with inflammation, or some combination thereof.

In one embodiment of the invention, the inflammation to be reduced maybe associated with an inflammation-related condition, as that term isutilized below. The inflammation may be a precursor of, a causativeeffect of, or a symptom of the inflammation-related condition. Inanother embodiment of the invention, the cause of the inflammation to bereduced may be unknown, and its relation to an inflammation-relatedcondition also unknown.

A compound may be determined to have reduced inflammation by monitoringthe symptoms of a patient exhibiting inflammation in one or more tissuesor organs. In one embodiment of the invention, a compound has reducedinflammation if the physiological indications of inflammation arereduced or the symptoms are lessened.

4. Neurotrophic Activity

In an embodiment, the invention includes methods of inducing release ofneurotrophic factors that exhibit neurotrophic effects on neurons. In afurther embodiment, the neurotrophic effects are on dopaminergicneurons. The invention further includes methods for mediating release ofneurotrophic factors from astroglia.

An embodiment of the invention provides methods for activatingneuronal-survival signaling pathways in a mammal or human subject inneed thereof that comprise administration of at least one compound ofthe invention. In a further embodiment, the compound is valproic acid(VPA) or salts thereof or valproate. In additional embodiment, thecompound comprises butyric acid and salts thereof. In other embodiment,the compound comprises 3-hydroxy-morphinan.

In an embodiment, the invention includes any of the methods forneuroprotection comprising methods for reducing inflammation describedabove in combination with methods for activating neuronal-survivalsignaling pathways.

In an embodiment, one or more compounds of the invention exhibit bothneurotrophic and neuroprotective activities. In a further embodiment,the compound is valproic acid.

In an embodiment, the invention includes methods for induction ofrelease of glial cell-line-derived neurotrophic factor (GDNF). GDNF isseveral orders of magnitude more potent than other neurotrophins. Afurther embodiment provides methods for induction of GDNF to promotesurvival and protection of nerve cells. A still further embodimentprovides methods including administration of at least one compound ofthe invention to induce release of GDNF. In a further embodiment, thecompound is valproic acid (VPA) or valproate.

In an embodiment, a method comprises administering an effective amountof an inhibitor of histone deacetylase to a cell or tissue that iscapable of producing glial-derived neurotrophic factor. In someembodiments, the tissue is nerve tissue comprising astroglial cells. Inother embodiments, the inhibitor of histone deacetylase comprisesvalproic acid, butyric acid and/or salts thereof. In some embodiments,an effective amount is about 0.1 to about 1 μM, more preferably about0.2 to about 0.8 μM, more preferably about 0.4 to about 0.6 μM.

In another embodiment, a method of providing a neurotrophic effectcomprises administering to a cell or tissue an effective amount of3-hydroxymorphinan. In some embodiments, the tissue or cells compriseastroglia cells. In some embodiments, an effective amount comprisesabout 1 to 10 μM, more preferably about 1 to about 5 μM, and morepreferably about 2.5 to 5 μM.

5. Treatment of Disorders or Conditions

One aspect of the invention includes methods of treating inflammation inthe brain characterized by activation of microglia and astroglia.Another aspect of the invention includes methods of treating Parkinson'sdisease, Alzheimer's disease, ALS, MS, atherosclerosis, diabetes,arthritis, sepsis, septic shock, endotoxemia, multiple organ failure, ororgan damage.

The invention also includes methods of treating an inflammation-relatedor other neurological condition that comprises administering aneffective amount of a peptide comprising an amino acid sequence GGF. Insome embodiments, a polypeptide or peptide comprises an amino acidsequence GGF (Gly-Gly-Phe) and is other than or excludes the full lengthsequence of dynorphin A (SEQ ID NO:1). Preferably, the polypeptide caninhibit the activity of NADPH oxidase and/or the generation of reactiveoxygen species. In other embodiments, a peptide comprising GGF has nomore than 16 amino acids, more preferably, about 3 to 16 amino acids,more preferably about 3 to 10 amino acids, and more preferably about 3to 5 amino acids. In other embodiments, a peptide that comprises GGF maybe chemically modified or linked to a heterologous polypeptide. Inpreferred embodiments, a peptide comprising GGF may be linked to amolecule or compound that enhances intracellular transport or transportacross the blood brain barrier.

In one embodiment of the invention, the effective amount of the compoundcan be from about 10⁻⁵ M to about 10⁻¹⁵ M. In another embodiment of theinvention, the effective amount of the compound is from about 10⁻⁵ M toabout 10⁻⁷ M, or about 10⁻¹³ M to about 10⁻¹⁵ M.

The invention also includes methods of treating an inflammation-relatedor other neurological condition that comprises administering aneffective amount of valproic acid (VPA), valproate, butyric acid, orother salts thereof. Valproic acid is a short chain fatty acid,previously used for treatment of bipolar disorders and seizures. In anembodiment, the therapeutically effective amount of valproic acid isfrom about 10⁻³ to about 10⁻⁶ M. In an embodiment of the invention, theeffective amount of valproic acid or salts thereof (e.g., valproate) isfrom about 0.35 to 1 mM.

In other embodiments, other compounds can be utilized to treatinflammation associated conditions and/or other neurological conditionssuch as Alzheimers, Parkinsons, multiple sclerosis, and ALS. Preferably,the compounds are those that can penetrate the blood brain barrier andact on inflammation in the brain. The other compounds comprise naloxone,naltrexone, dextromethorphan, an opioid peptide such as Met enkephalinor Leu-enkephalin, or mixtures thereof. In some embodiments thecompounds can be administered in an ultra low concentration, so as toachieve a concentration of about 10⁻¹³ M to about 10⁻¹⁵ M. In anembodiment, ultra low concentrations comprise about 10 pg/kg to about1000 pg/kg, more preferably about 1000 pg/kg, more preferably about 100pg/kg, and more about preferably 10 pg/kg.

As used herein, the word “treating” includes, but is not limited to,alleviating or reliving symptoms associated with the disease; inhibitingthe progression of the disease, i.e., arresting its development;lessening the likelihood of the occurrence of the disease; reversing orlimiting or lessening the deleterious effects of the disease on thediseased and related tissue.

As used herein, the phrase “inflammation-related condition” includes anydisease, disorder, or condition that is caused by or related to aninflammatory process within one or more tissue or serum of the body of amammal. In another embodiment, an “inflammation-related condition”includes inflammation-related diseases that are caused by or related toan inflammatory process within neurological tissue. Examples ofinflammation-related condition that are caused by or related to aninflammatory process within neurological tissue include, but are notlimited to Alzheimer's disease, Parkinson's disease, amytrophic lateralsclerosis (ALS), and multiple sclerosis (MS). Examples ofinflammation-related condition that are caused by or related to aninflammatory process within tissues other than neurological tissuesinclude, but are not limited to atherosclerosis, diabetes, arthritis,sepsis, septic shock, endotoxemia, multiple organ failure,cardiovascular disease, and organ damage, such as liver damage forexample.

Included in the invention are methods of treating Alzheimer's disease,Parkinson's disease, ALS, MS, atherosclerosis, diabetes, arthritis,sepsis, septic shock, endotoxemia, multiple organ failure, and organdamage that comprise administering an ultra-low dose of at least one ofthe compounds of the invention to a mammal.

The mammal to be treated may be already diagnosed with theinflammation-related condition, may be at risk of developing theinflammation-related condition, may have experienced a trauma that mayincrease the chances of the inflammation-related condition occurring, ormay have no heightened risk of developing the inflammation-relatedcondition.

6. Methods of Identifying Therapeutic Targets

The invention also includes methods of identifying compounds that may beeffective in treating an inflammation-related condition that comprisecontacting NADPH oxidase or a solution containing the gp91 subunit ofNADPH oxidase with a candidate compound, and determining whether thecandidate compound inhibits NADPH oxidase as compared to NADPH oxidasein the absence of the compound, wherein a compound may betherapeutically effective in treating an inflammation-related conditionif the compound decreases the activity of the NADPH oxidase or its gp91subunit.

The invention also includes methods of identifying compounds that may beeffective in treating an inflammation-related condition that comprisemonitoring the behavior of the gp91 subunit NADPH oxidase and/orcontacting the NADPH oxidase or a solution containing NADPH oxidase witha compound, monitoring the effect of the compound on the activity of thegp91 subunit NADPH oxidase, and comparing that effect with the activitygp91 subunit of the NADPH oxidase without the compound, wherein acompound may be therapeutically effective in treating aninflammation-related condition if the compound decreases the activity ofthe gp91 subunit NADPH oxidase.

The invention also includes methods of identifying compounds that may beeffective in treating an inflammation-related condition that comprisecontacting the innate immune cell with a compound, and comparing thateffect in the presence of the compound with the activity, oroveractivity of an innate immune cell without the compound, wherein acompound may be therapeutically effective in treating aninflammation-related condition if the compound decreases the activity,or overactivity of the innate immune cell.

The monitoring steps referred to in the methods of identifying targetscan be accomplished by monitoring one or more of the pro-inflammatoryfactors discussed above.

7. Compounds of the Invention

It has previously been shown that dynorphin A (DYNA (1-17)Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln (SEQID NO:1) a kappa receptor agonist, protects mesencephalic dopaminergicneurons from microglia-mediated neurotoxicity.

It was unexpectedly discovered that the minimal, and novel fragmentglycine-glycine-phenylalanine (GGF) (SEQ ID NO. 2), can be used toeffectuate methods of the invention. The observation that thisparticular peptide fragment could be utilized was surprising because asseen above, it does not need the initial amino acid of dynorphin A,which was commonly thought necessary for binding to the active site ofthe Kappa receptor. In one aspect of the invention, a peptide comprises,or consists of an amino acid sequence GGF. In some embodiments, apolypeptide or peptide comprises an amino acid sequence GGF(Gly-Gly-Phe) and is other than or excludes the full length sequence ofdynorphin A (SEQ ID NO:1). Examples of such peptides are provided inTable 1. Preferably, the polypeptide can inhibit the activity of NADPHoxidase and/or the generation of reactive oxygen species. In otherembodiments, a peptide comprising GGF has no more than 16 amino acids,more preferably, about 3 to 16 amino acids, more preferably about 3 to10 amino acids, and more preferably about 3 to 5 amino acids. In otherembodiments, a peptide that comprises GGF may be chemically modified orlinked to a heterologous polypeptide. In preferred embodiments, apeptide that comprises GGF may be linked to a molecule or compound thatenhances intracellular transport or transport across the blood brainbarrier.

Other compounds that can be used in the invention include morphinans.Exemplary morphinans include, but are not limited to naloxone,naltrexone, dextromethorphan. Opioid peptides including {Met5}-enkephalin, and {Leu 5}-enkephalin can also be used in the invention.In one embodiment of the invention, either naloxone or dextromethorphanare used in methods of the invention.

Naloxone is commonly known as Narcan, and refers to the chemicalcompound: (5α)-4,5-Epoxy-3,14-dihydroxy-17-(2-propenyl)morphinan-6-one,or 17-allyl-4,5α-epoxy-3,14-dihydroxymorphinan-6-one. The structure ofwhich is given below.

Dextromethorphan (DM), which refers to the compound,d-3-methoxy-N-methylmorphinan, is commonly used as an antitussive, andis commercially available in Robitussin® and Sucrets®. The structure ofdextromethorphan is given below.

In various embodiments, methods of the invention utilize ultra lowamounts, dosages, or concentrations of one or more compounds of theinvention. As used herein, the phrase “ultra low” refers toconcentrations between and inclusive of about 10⁻¹³ M to 10⁻¹⁵ molar ormoles/liter (“M”). In one embodiment, compounds of the invention areutilized in concentrations between and inclusive of about 10⁻¹³ M to10⁻¹⁴ M. In another embodiment of the invention, compounds of theinvention are utilized in concentrations between and inclusive of about10⁻¹⁴ M. In an embodiment, ultra low concentrations comprise about 10pg/kg to about 1000 pg/kg, more preferably about 1000 pg/kg, morepreferably about 100 pg/kg, and more about preferably 10 pg/kg.

The novel tripeptide fragment, GGF and peptides comprising GGF, has atleast two ranges at which it is “effective” in methods of the invention.GGF can be used at concentrations of about 10⁻⁵ to about 10⁻⁷ M, orabout 10⁻¹³ to about 10⁻¹⁶ M.

Valproic acid (VPA), a simple eight-carbon branched-chain fatty acid.VPA is available either as the free acid, or in a salt form. One saltform of VPA is sodium valproate. Butyric acid is a four-carbon fattyacid. Butyric acid is available as a free acid, or in a salt form, forexample Sodium Butyrate. Valproic acid, sodium butyrate and relatedcompounds are effective in neurotrophic methods of the invention atconcentrations from about 0.35 to 1 mM.

Compounds of the invention can be prepared by any method known to thoseof skill in the art, having read this specification. Furthermore,compounds of the invention are commercially available through a numberof different sources. For example, the tripeptide, GGF, can be obtainedfrom BACHEM, (Torrance Calif.).

Valproic acid is available from Sigma-Aldrich (St. Louis, Mo.).

Salts

Some of the compounds of the invention may be capable of forming bothpharmaceutically acceptable acid addition and/or base salts. Base saltsare formed with metals or amines, such as alkali and alkaline earthmetals or organic amines. Examples of metals used as cations are sodium,potassium, magnesium, calcium, and the like. Also included are heavymetal salts such as, for example, silver, zinc, cobalt, and cerium.Examples of suitable amines are N,N′-dibenzylethylenediamine,chloroprocaine, choline, diethanolamine, ethylenediamene,N-methylglucamine, and procaine.

Pharmaceutically acceptable acid addition salts are formed with organicand inorganic acids. Examples of suitable acids for salt formation arehydrochloric, sulfuric, phosphoric, acetic, citric, oxalic, malonic,salicylic, malic, gluconic, fumaric, succinic, ascorbic, maleic,methanesulfonic, and the like. The salts are prepared by contacting thefree base form with a sufficient amount of the desired acid to produceeither a mono or di, etc. salt in the conventional manner. The free baseforms can be regenerated by treating the salt form with a base. Forexample, dilute solutions of aqueous base can be utilized. Diluteaqueous sodium hydroxide, potassium carbonate, ammonia, and sodiumbicarbonate solutions are suitable for this purpose. The free base formsdiffer from their respective salt forms somewhat in certain physicalproperties such as solubility in polar solvents, but the salts areotherwise equivalent to their respective free base forms for thepurposes of the invention.

One example of a pharmaceutically acceptable salt includes ahydrochloride salt of a compound of the invention.

8. Compositions and Administration Methods

The compounds of the present invention can be formulated aspharmaceutical compositions and administered to a mammalian host,including a human patient, in a variety of forms adapted to the chosenroute of administration.

The compounds are preferably administered in combination with apharmaceutically acceptable carrier, and can be combined with orconjugated to specific delivery agents, including targeting antibodiesand/or cytokines.

The compounds can be administered by known techniques, such as orally,parentally (including subcutaneous injection, intravenous,intramuscular, intrasternal or infusion techniques), by inhalationspray, topically, by absorption through a mucous membrane, or rectally,in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants or vehicles.Pharmaceutical compositions of the invention can be in the form ofsuspensions or tablets suitable for oral administration, nasal sprays,creams, sterile injectable preparations, such as sterile injectableaqueous or oleagenous suspensions or suppositories.

For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents, andlubricants known in the art.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons, or othersolubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

Solutions or suspensions of the compounds can be prepared in water,isotonic saline (PBS), and optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethylene,glycols, DNA, vegetable oils, triacetin and mixtures thereof. Underordinary conditions of storage and use, these preparations can contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage form suitable for injection or infusion usecan include sterile, aqueous solutions, dispersions, or sterile powderscomprising an active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions.The final dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol such as glycerol, propylene glycol, or liquidpolyethylene glycols, and the like, vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size, in the case of dispersion, orby the use of nontoxic surfactants. The prevention of the action ofmicroorganisms can be accomplished by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be desirable toinclude isotonic agents, for example, sugars, buffers, or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the inclusion in the composition of agents delayingabsorption such as, for example, aluminum monosterate hydrogels andgelatin.

Sterile injectable solutions are prepared by incorporating theconjugates in the required amount in the appropriate solvent withvarious other ingredients as enumerated above and, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying techniques, which yielda powder of the active ingredient plus any additional desired ingredientpresent in the previously sterile-filtered solutions.

The dose of the compound to be administered can depend at least in partupon the patient, the patient's medical history, and the severity of thedisease or disorder. Dosages for adult humans may range from betweenabout 10 pg/kg to 1 mg/kg. In another embodiment of the invention, thedosage for an adult human may range from about 10 pg/kg to about 1mg/kg. These doses may be repeated up to several times per day. Inaddition, lower and higher doses may be more appropriate depending onthe individual patient and the disease or condition to be treated.

WORKING EXAMPLES

The following examples provide nonlimiting illustrations of variousembodiments of the invention.

Cell culture ingredients were obtained from Invitrogen (Carlsbad,Calif.). [³H]Dopamine (DA, 30 Ci/mmol) was purchased from PerkinElmerLife Sciences (Boston, Mass.). The monoclonal antibody against the CR3complement receptor (OX-42) was obtained from BD PharMingen (San Diego,Calif.). The polyclonal anti-tyrosine hydroxylase (TH) antibody was agenerous gift from Dr. John Reinhard (GlaxoSmithKline, Research TrianglePark, N.C.) (The antibody is also commercially available.). Themonoclonal antibody against the CR3 complement receptor (OX-42) wasobtained from BD PharMingen (San Diego, Calif.). The Vectastain ABC kitand biotinylated secondary antibodies were purchased from VectorLaboratories (Burlingame, Calif.). The CyQUANT cell proliferation assaykit was purchased from Molecular Probes, Inc. (Eugene, Oreg.). GriessReagent is available from Promega Corporation (Madison, Wis.).

NADPH oxidase-deficient (gp91phox^(−/−)) and wild-type C57BL/6J(gp91phox^(+/+)) mice were obtained from The Jackson Laboratory (BarHarbor, Me.). Breeding of the mice was performed to achieve timedpregnancy with the accuracy of ±0.5 d. Timed-pregnant Fisher F344 ratswere obtained from Charles River Laboratories (Raleigh, N.C.). Housingand breeding of the animals were performed in strict accordance with theNational Institutes of Health guidelines.

Data are presented as the mean ±S.E.M. for multiple comparisons ofgroups using ANOVA. Statistical significance between groups was assessedby paired or unpaired Student's t-test, with Bonferroni's correction. Avalue of p<0.05 was considered statistically significant.

Example 1 Femtomolar Concentrations Of DM Protect LPS-InducedDopaminergic Neurodegeneration

In order to explore whether DM at femtomolar concentrations isneuroprotective against inflammation-mediated dopaminergic neurondegeneration, a wide range of concentrations of DM (10⁻⁵ M to 10⁻¹⁷ M)were tested.

Neuron-glia cultures were prepared from the ventral mesencephalictissues of embryonic day 13-14 Fisher F344 rats or day 12-13 wild-typeC57BL/6J (gp91phox^(+/+)) mice. Dissociated cells were seeded at1×10⁵/well and 5×10⁵/well to poly-D-lysine-coated 96-well and 24-wellplates, respectively. Cells were maintained at about 37° C. in ahumidified atmosphere of 5% CO₂ and 95% air, in minimal essential medium(MEM) containing 10% fetal bovine serum (FBS), 10% horse serum (HS), 1gm/1 glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μMnonessential amino acids, 50 U/ml penicillin, and 50 μg/ml streptomycin.Seven-day-old cultures were used for treatment. At the time oftreatment, immunocytochemical analysis indicated that the ratneuron-glia cultures were made up of 11% microglia, 48% astrocytes, 41%neurons, and 1% tyrosine hydroxylase-immunoractive (TH-IR) neurons. Thecomposition of the neuron-glia cultures of NADPH oxidase-deficient micewas very similar to that of the wild-type mice in that there were 12%microglia, 48% astrocytes, 40% neurons, and 1% TH-IR) neurons.

Dextromethorphan (obtained from Sigma-Aldrich (St. Louis, Mo.) asdextromethorphan hydrobromide) was freshly prepared as a stock solution(1 mM) in ddH₂O and sterile-filtered right before use. For treatment ofthe cultures, the DM was serially diluted (10×) with fresh culturemedium containing 2% of fetal bovine and horse serum. The neuron-gliacultures were pretreated with 10⁻⁵-10⁻¹⁷ M DM 30 minutes prior totreatment with 10 ng/ml of LPS.

Seven days after treatment, the degeneration of dopaminergic neurons wasassessed by [³H]-dopamine (DA) uptake assays. The [³H]-DA uptake assayswere performed as follows. Cultures were washed twice with warmKrebs-Ringer buffer (KRB, 16 mM sodium phosphate, 119 mM NaCl, 4.7 mMKCl, 1.8 mM CaCl₂, 1.2 mM MgSO₄, 1.3 mM EDTA, and 5.6 mM glucose; pH7.4)and then incubated for about 20 minutes at about 37° C. with 1 μM[³H]-DA in KRB in order to allow for the uptake of DA. Afterwards, thecultures were washed (three times) with ice-cold KRB and the cells werecollected in 1 N NaOH. Radioactivity was determined by liquidscintillation counting. Nonspecific DA uptake observed in the presenceof mazindol (10 μM) was subtracted as a control.

The results are expressed as a percentage of the control cultures andare the mean ±S.E.M of three to six individual experiments withtriplicates in each experiment. ##, P<0.01 compared with the controlculture; *, P<0.05 compared with the LPS-treated culture.

As shown in FIG. 1, DM at micromolar (10⁻⁵ and 10⁻⁶ M) concentrationsattenuated the LPS-induced decrease in [³H]-dopamine uptake. However, itwas surprising that femtomolar (10⁻¹³ M and 10⁻¹⁴ M) concentrations ofDM showed equipotent neuroprotective effect as that of DM at micromolarconcentrations. It is interesting to note that nanomolar and picomolarconcentrations of DM (10⁻⁸ M to 10⁻²M) showed no protective effects. Itappears that this dose-response curve can be divided into threeregions: 1) micromolar responsive region, 2) non-response region and 3)femtomolar responsive region. Thus, 10⁻5 M, 10⁻¹⁰ M and 10⁻¹⁴M wereselected as representative concentrations of each of the three regionsfor further study.

Example 2 Morphological Analysis of DM-Elicited Neuroprotection

The degeneration of dopaminergic neurons was assessed by the observationof changes in tyrosine hydroxylase-immunoreactivity (TH-IR) neuronmorphology and a count of the number of TH-IR neurons in neuron/gliacultures prepared and treated with LPS and 10⁻⁵ M, 10⁻¹⁰ M, and 10⁻¹⁴ MDM as in Example 1 (counting was performed in a double-blind manner bythree individuals).

Dopaminergic neurons were recognized with the anti-TH antibody andmicroglia were detected with the OX-42 antibody, which recognizes theCR3 receptor. This was accomplished as follows: 3.7% formaldehyde-fixedcultures were treated with 1% hydrogen peroxide for about 10 minutesfollowed by sequential incubation with a blocking solution (30 min),primary antibody (overnight, 4° C.), biotinylated secondary antibody (2hours), and ABC reagents (40 min). The color was developed with3,3′-diaminobenzidine.

For morphological analysis, the images were recorded with an invertedmicroscope (Nikon, Tokyo, Japan) connected to a charge-coupled devicecamera (DAGE-MTI, Michigan City, Ind.) operated with the MetaMorphsoftware (Universal Imaging Corporation, Downingtown, Pa.). For visualcounting of TH-IR neurons, three wells with the same treatment in the24-well plate were counted under the microscope at 100× magnification bythree different individuals. The average of these scores was reported.

FIG. 2A shows the results as a percentage of the control cultures, andare the mean ±S.E.M of five individual experiments. ##, P<0.01 comparedwith the control culture. *, P<0.05 compared with the LPS-treatedculture. As seen there, treatment with 10 ng/ml LPS alone caused asignificant reduction in the loss of TH-IR neurons (60%) compared withvehicle-treated control cultures. Thirty minute pretreatment with DM10⁻⁵ and 10⁻¹⁴ M significantly attenuated the LPS-induced reduction inthe number of TH-IR neurons by 37 and 28%, respectively. DM at 10⁻¹⁰ Mhad no protective effects on dopaminergic neuron degeneration. Theresults from the cell counts (FIG. 2A) were comparable to that of the[³H]-dopamine uptake study of Example 1 (FIG. 1).

Immunocytochemical analysis, shown in FIG. 2B shows the loss of theintricate dendrite network in the LPS-treated group. A more elaboratedendrite network was observed in 10⁻⁵ M and 10⁻¹⁴ M DM-treated groups,while DM 10⁻¹⁰ M failed to show improvement. This observation is alsoconsistent with the DA uptake and neuron numeration analysis.

Example 3 Femtomolar Concentrations of DM Protect Aβ-InducedDopaminergic Neurodegeneration

The neuroprotective effects of DM both at micro- and femtomolarconcentrations against Aβ-induced neurotoxicity were also investigated.

Neuron-glia co-cultures were prepared and treated with vehicle alone, Aβ0.75 μM alone, or DM 30 min prior to treatment with Aβ 0.75 μM similarto Example 1. Amyloid-β peptide (25-35 and 1-42) was obtained fromAmerican Peptide Co., Inc (Sunnyvale, Calif.). Neurotoxicity wasassessed by DA uptake as in Example 1. Results are expressed as apercentage of the control cultures and are the mean ±S.E.M. of three toeight individual experiments with triplicates in each experiment. ##,P<0.01 compared with the control culture; *, P<0.05 compared withAβ-treated culture.

Results are shown in FIG. 3, where the neuroprotective effect of DM atboth micro- and femtomolar concentrations can be seen against Aβ-inducedneurotoxicity.

Example 4 Protective Effect of DM in Aβ- or 1-methyl-4-phenylpyridinium(MPP⁺)-Induced Dopaminergic Neurodegeneration

Neuron-enriched culture were prepared from the ventral mesencephalictissues of embryonic day 13-14 Fisher F344 rats (Charles RiverLaboratories, Raleigh, N.C.) as follows. Dissociated cells were seededat 1×10⁵/well in 96-well and 5×10⁵/well to poly-D-lysine-coated 96-welland 24-well plates, respectively. Glial proliferation was suppressed bythe inclusion of cytosine β-D-arabinocide (5-10 μM). Seven day oldcultures were used for treatment, which were composed of 91% neurons, 9%astrocytes, and <0.1% microglia. The cultures were pre-treated with OM,10⁻⁵ M, 10⁻¹⁰ M, and 10⁻¹⁴M DM 30 minutes before treatment with vehiclealone, 4 μM Aβ or 0.5 μM MPP⁺⁵ as in Example 1. DA uptake was alsomeasured as in Example 1 and the results are expressed in both FIGS. 4Aand B as a percentage of the control cultures and are the mean ±S.E.M.of four individual experiments with triplicates in each experiment.

As shown in FIG. 4A, 9 days after treatment with 4 μM Aβ (1-42), DAuptake was reduced by 50% compared with the control cultures.Pretreatment of the neuronal cultures with DM (10⁻⁵ M, 10⁻¹⁰M, and 10⁻¹⁴M) before Aβ (1-42) treatment did not significantly alter the magnitudeof the Aβ (1-42)-induced reduction of DA uptake in the cultures. Asimilar effect was observed in the samples treated with MPP⁺ (FIG. 4B).These results suggested that the presence of glial cells may benecessary for DM to express its neuroprotective effect.

In comparing the results from the above examples, the protective effectis only observed in neuron-glia cultures, but not in neuron-enrichedcultures, since the DM failed to show a protective effect against Aβ- orMPP⁺-induced dopaminergic neurotoxicity in neuron-enriched cultureregardless of the concentration of DM. This comparison may indicate thatfemtomolar DM-elicited protection against inflammation-mediateddopaminergic neurotoxicity is dependent on the presence of microglia.

Example 5 Femtomolar DM Inhibits LPS-Induced Microglia Activation

LPS can activate microglia to overproduce pro-inflammatory cytokines andfree radicals, such as NO, PGE₂, TNFα, superoxide, and other reactiveoxygen species (ROS), which in turn can cause neurodegeneration.

Neuron-glia cultures were prepared and treated with vehicle, LPS (5ng/ml), or LPS plus DM respectively as in Example 1. Twelve hours later,cultures were immunostained with anti-OX-42 antibody. Images shown arerepresentative of three separate experiments. The immunostaining andmorphological analysis was accomplished as in Example 2.

As shown in FIG. 5, LPS treatment transformed the resting, round shapeof microglia into the enlarged, irregular shape of activated microglia.Pre-treatment with DM at 10⁻⁵ M and 10⁻¹⁴ M prevented the LPS-inducedactivation of microglia. In contrast, DM at 10⁻¹⁰ M didn't significantlyinhibit microglia activation by LPS.

FIG. 5 depicts photomicrographs of microglia showing the inhibitoryeffect of DM on LPS-induced microglial activation.

Different pro-inflammatory factors that are released from microglia werealso monitored. The production of NO was determined by measuring theaccumulated levels of nitrite in the supernatant with the Griessreagent, and release of TNFα was measured with a rat TNFα enzyme-linkedimmunosorbent assay kit from R & D System (Minneapolis, Minn.).

PGE₂ in supernatant was measured with a prostaglandin E₂ EIA kit fromCayman (Ann Arbor, Mich.) according to the manufacturer's instructions.

The release of superoxide was determined by measuring the superoxidedismutase (SOD)-inhibitable reduction of cytochrome c. To measure theimmediate release of superoxide from microglia-enriched or neuron-gliaafter stimulation, cultures grown in 96-well plates were switched tophenol red-free HBSS (50 μl/well). To each well was added 50 μl of HBSScontaining vehicle or DM. The cultures were then incubated at about 37°C. for about 30 min followed by 50 μL of ferricytochrome c (100 μM) inHBSS, with and without 600 U/ml SOD, 50 μl of vehicle or LPS in HBSS.The absorbance at 550 nm was read with a SpectraMax Plus microplatespectrophotometer (Molecular Devices, Sunnyvale, Calif.).

The production of intracellular reactive oxygen species was measured byDCFH oxidation. The DCFH-DA (Molecular Probes, Eugene, Oreg.) reagentpassively diffuses into cells in which it is hydrolyzed by intracellularesterase to liberate 2′-7′-dichlorofluoressein which, during reactionwith oxidizing species, yields a highly fluorescent compound2′-7′-dichlorofluorescein (DCF) that is trapped inside the cell. Foreach measurement, a fresh stock solution of CM-H2-DCFDA (5 mM) wasprepared in dimethylsulfoxide. CM-H2-DCFDA, diluted to a finalconcentration of 1 μM in phenol red-free HBSS containing 2% FBS and 2%HS, was added to cultures and incubated for about 30 min at about 37° C.After washing two times with warm HBSS, vehicle or stimulators in HBSSwere added to cultures. After incubation for about 30 min at about 37°C., fluorescence intensity was measured at 485 nm for excitation and 530nm for emission using a SpectraMax Gemini XS fluorescence microplatereader (Molecular Devices).

Results are expressed in FIGS. 6A, 6B, and 6C as a percentage of the LPScultures, in 6D as a percentage of control, and in 6E as absorbancedifference above control value. The results are the mean ±S.E.M. of fourindividual experiments with triplicates in each experiment. *, P<0.05compared with LPS culture.

As seen in FIGS. 6A-6C, pre-treatment with DM at 10⁻⁵ M and 10⁻¹⁴ Msignificantly decreased the LPS-induced increase in the release of NO,PGE₂, TNFα,(FIG. 6C), superoxide (FIG. 6D), and intracellular reactiveoxygen species (FIG. 6E) whereas DM at 10⁻¹⁰ M showed no significantreduction of any of the species.

Example 6 Role of ROS in Mediating DM-Elicited Neuroprotective Effect

To further study the role of ROS in DM-elicited neuroprotection,neuron-glia cultures were prepared from NADPH oxidase-deficient(PHOX^(−/−)) and wild-type (PHOX^(+/+)) mice.

Microglia were prepared from the whole brains of 1-day-old Fisher F344rats or NADPH oxidase-deficient (gp91phox^(−/−)) (Jackson Laboratory,Bar Harbor Me.) or wild-type mice (C57 BL/6J (gp91phox^(+/+)) (JacksonLaboratory, Br Harbor, Me.), as in the above examples.Immunocytochemical analysis accomplished as above indicated that thecultures were 95-98% pure for microglia. Cells were seeded at 1×10⁵/wellin 96-well plates and used for treatment the following day.

The neuron-glia cultures were treated with vehicle, LPS 10 ng/ml alone,and DM (10⁻¹⁴ M) 30 min pretreatment followed by LPS treatment as inExample 1. Neurotoxicity was assessed by DA uptake as described inExample 1. TNFα production was measured by ELISA and iROS was determinedby DCFDA as in Example 5. Results are expressed as a percentage of thecontrol culture in FIG. 7A, pg/ml in FIG. 7B, and difference fromcontrol in FIG. 7C, respectively, and are the mean ±S.E.M. of fiveindividual experiments with triplicates in each experiment. *, P<0.05compared with LPS culture.

As shown in FIG. 7A, in neuron-glia cultures prepared from PHOX^(+/+)mice, LPS treatment reduced [³H]-dopamine uptake by 46%; 10⁻¹⁴Msignificantly attenuated this decrease. In contrast, LPS treatmentreduced the uptake capacity by only 25% in PHOX^(−/−) mice and DM(10⁻¹⁴M) failed to show any protective effect. Similar to DA uptakeresult, LPS-induced iROS production in PHOX^(−/−) mice is only half ofthat in PHOX^(+/+) mice, and DM at 10⁻¹⁴ M significantly inhibited iROSproduction in PHOX^(+/+) mice, while failed to show any effect inPHOX^(−/−) mice (FIG. 7C). Consistently, LPS-induced TNFα production inPHOX^(−/−) mice is two thirds of that in PHOX^(+/+) mice, and DM at10⁻¹⁴ M was able to significantly reduce TNFα production, which was notseen in PHOX^(−/−) mice (FIG. 7B). These results strongly support thepossibility that inhibition of ROS production and subsequently TNFαproduction may be associated with the neuroprotective effect of DM atfemtomolar concentrations.

PHOX is the major superoxide-producing enzyme in microglia and the majorcontributor to the increase in iROS concentrations in response to avariety of immune stimulants such as LPS, β-amyloid peptides (Aβ). Forexample, through the activation of PHOX, Aβ at low concentrationsincreases the production of neurotoxic superoxide, but not the otherfactors, such as nitrite and TNFα. The findings that micromolar andfemtomolar concentrations of DM could protect Aβ-induced dopaminergicneurotoxicity may suggest that DM affords its neuroprotection byinhibiting PHOX activity. Femtomolar DM, while significantly lesseningthe LPS-induced DA uptake reduction in wild-type mice, has nosignificant protective effect in PHOX^(−/−) mice (FIG. 7A). Theseobservations support the contention that the protective effect offemtomolar DM may be mediated through the inhibition of PHOX activity.Activation of PHOX in microglia not only increases the production ofsuperoxide, but also indirectly increases the intracellular ROSconcentration, possibly through the conversion of superoxide to H₂O₂,which is membrane permeable. Increase of iROS can intensify theactivation of NFκB, which leads to higher TNFα, PGE₂ production. Theresult that femtomolar DM inhibited TNFα production in wild type whilenot in PHOX^(−/−) mice further supports the notion that femtomolar DMmay be acting on PHOX.

Example 7 Effect of Post-Treatment with DM on LPS-Induced DopaminergicNeurodegeneration

This example tests whether or not post-treatment with DM is stillneuroprotective in LPS-induced neurotoxicity. Neuron-glia co-cultures asprepared in Example 1 were first treated with LPS (20 ng/ml) for twelvehours, and then LPS was removed by removing the media from the culturesand washing twice with PBS.

Different concentrations of DM (10⁻⁵, 10⁻¹, and 10⁻¹⁴ M) were then addedto the cultures and incubation was continued for another 6 or 7 days.The DA uptake and the superoxide production were measured as in Example1 and 5. Results are expressed as a percentage of the control culturesand are the mean ±S.E.M of three to six experiments with triplicates ineach experiment. ##, P<0.01 compared with control culture; *, P<0.05compared with the LPS-treated culture.

As seen in FIG. 8A, the presence of LPS in the media for only 12 hourswas capable of reducing the dopamine uptake capacity by 70%.Post-treatment with DM showed a protective effect at concentrations of10⁻⁵ and 10⁻¹⁴ M, but not at 10⁻¹⁰ M. In the same experiment, superoxidelevels were measured in companion cultures 24 hours after LPS treatment.Consistent with pre-treatment studies, post-treatment with DM at 10⁻⁵and 10⁻¹⁴ M concentrations significantly inhibited LPS-induced increasein superoxide production. In contrast, neither pre-treatment norpost-treatment with DM at 10⁻¹⁰ M significantly affected the productionof superoxide, as seen in FIG. 8B.

Example 8 Determination of Possibility of Direct Action of DM on iNOSand COX 2 Enzymes

Western blots were used to analyze possible effects of DM on iNOS andCOX2 production in rat microglia enriched cultures. The cultures wereprepared and treated with vehicle, 0M DM, 10⁻¹⁴DM, LPS alone, and LPSwith different concentrations of DM. The production of iNOS (induciblenitric oxide synthenase) and COX2 (cycloxyase 2) in the culture extractswere detected by Western Blot assay. Protein levels of iNOS werequantified by a densitometer system (n=3) and are reported in FIG. 9B.Data in FIG. 9B represent the mean ±S.E.

Our speculation that femto-molar DM could reduce NO and PGE₂ productionby directly acting on enzymes iNOS and COX2 was strongly supported bythe observation that femto-molar DM 30 min pretreatment reduced NO andPGE₂ production while failing to affect the protein content of iNOS andCOX2 (FIG. 9).

After the LPS-LBP complex is bound to the membrane protein CD14/TLR4,NFκB is triggered through cascading signaling pathway to regulate themRNAs encoding iNOS and COX2, which produce NO and PGE₂ respectively.

Example 9 Effect of Post Treatment DM on LPS-Treated Micro-Glia Cultures

The cultures were prepared as in Example 1 and were first treated withLPS 20 ng/ml or vehicle; 12 hours later the LPS was removed and thecultures were treated with 10⁻⁵ M, 10⁻¹⁰ M, and 10⁻¹⁴ M DM. Twenty fourhours later, nitrite oxide and PGE₂ production were assessed as inExample 5. The results are expressed as a percentage of LPS treatedculture and are the mean ±S.E.M of four and three experiments withtriplicates in each experiment. *, p<0.05 compared with the LPS-treatedculture. ###, P<0.001 compared with the control culture.

FIGS. 10A and 10B demonstrate that femtomolar DM post treatmentfollowing removal of LPS after 12 hours LPS treatment on neuron/gliaculture resulted in reduction of NO and PGE₂ production withoutaffecting iNOS and COX2 protein levels as evidenced by FIG. 9. SinceiNOS and COX2 accumulated within the 12 hours of LPS treatment aresufficient for continuing the production of NO and PGE₂ even in theabsence of LPS, it was concluded that DM decreased NO and PGE₂production by directly inhibiting the activities of these enzymes.Reduction in the production of nitrite, PGE₂ and TNFα, together with thedrastic suppression of ROS production, is thought to be one of themechanisms underlying the potent neuroprotective effect of femto-molarDM.

Example 10 Effect of Various Peptides on LPS-Induced DopaminergicNeurodegeneration

Femtomolar concentrations of several small peptide fragments of varyinglengths and sequences were tested for their ability to protect DAneurons from LPS-induced neurodegeneration in vitro.

Neuron-glia cell cultures were prepared as in Example 1, and pretreatedwith the various peptide fragments of Table 1 (peptide fragments wereobtained from BACHEM) for 30 minutes followed by addition of 5 ng/ml ofLPS. DA neurotoxicity was measured as an Example 1 at 7 days posttreatment.

The data in Table 1 are expressed as the percent of the control culturesand are the mean ±SEM of 3 experiments performed in triplicate. *P<0.05,**P<0.01, compared to control.

The Dyn A (2-4) peptide, glycine-glycine-phenylalanine (GGF), was foundto be the minimal peptide sequence required for neuroprotection, wherescrambling the sequence (GFG) proved to be ineffective.

TABLE 1 LPS LPS + Peptide LPS + Peptide LPS + Peptide Peptide Control (5ng/ml) 10⁻¹⁵ 10⁻¹⁴ 10⁻¹³ DynA 1-17 100 ± 3.9 40.1 ± 3.2 51.0 ± 9.4* 58.0± 6.5** 52.1 ± 6.2* DynA 2-17 100 ± 3.9 40.1 ± 3.2 50.2 ± 6.9* 62.7 ±8.0** 44.3 ± 8.2 DynA 1-5 100 ± 3.9 40.1 ± 3.2 54.5 ± 4.6* 63.5 ± 3.2**51.4 ± 4.1* DynA 2-5 100 ± 3.9 40.1 ± 3.2 50.5 ± 5.3* 58.9 ± 3.9** 59.4± 9.0* DynA 3-8 100 ± 3.9 40.1 ± 3.2 40.9 ± 5.9 43.5 ± 1.5 39.8 ± 6.2DynA 6-17 100 ± 3.9 40.1 ± 3.2 38.4 ± 3.9 41.9 ± 2.7 39.6 ± 5.3 DynA 1-3100 ± 3.9 40.1 ± 3.2 55.3 ± 3.54* 70.3 ± 3.38**   62 ± 3.73** GFG 100 ±3.9 40.1 ± 3.2 40.5 ± 7.3 37.1 ± 7.4 35.8 ± 5.6 GG 100 ± 3.9 40.1 ± 3.240.2 ± 5.8 42.6 ± 4.8 38.9 ± 4.6 GF 100 ± 3.9 40.1 ± 3.2 40.2 ± 6.3 41.3± 4.7 40.6 ± 5.5 Dynorphin A: YGGFLRRIRPKLKWDNQ

Example 11 Effect of Femtomolar Concentrations of Naloxone and GGF onLPS-Induced Dopaminergic Neurons

Mesencephalic neuron-glia cultures were prepared as in Example 1 andwere treated with either vehicle, LPS (5 ng/ml) or were pretreated for30 minutes with naloxone or GGF (10⁻¹²−10⁻¹⁶M) followed by addition ofLPS (5 ng/ml) as described in Example 1. DA neurotoxicity was measuredat 7 days post treatment as in Example 1. Dopaminergic neuronal deathwas determined at 7 days post treatment using immunocytochemicalstaining as in Example 2. The ability of GGF and naloxone to protect DAneurons from LPS-induced damage is depicted by immunocytochemicalanalysis with anti-TH antibody as in Example 2. The data are expressedas the percentage of the control cultures and are the mean ±SEM fromthree independent experiments, each performed with triplicate samples. *P<0.05, ** P<0.01 compared to control.

FIG. 11A shows that both the peptide GGF and naloxone exhibit similarneuroprotective qualities at femtomolar concentrations. The ability ofDA neurons in mesencephalic cultures to take up [³H] DA after exposureto LPS was enhanced by approximately 35% with 30 minute pretreatment ofeither naloxone or GGF, with the greatest level of protection conferredat the concentration of 10⁻¹⁴M for both compounds.

FIG. 11C shows that both 10⁻¹⁴M naloxone and 10⁻¹⁴M GGF protected THimmuno-reactive neurons from LPS-induced damage, such as loss ofdendrites, axon disintegration, and loss of DA neurons. FIG. 11Bevidences that GGF and naloxone pretreatment protected againstLPS-induced DA neuron cell loss, with the peak protection occurring at10⁻¹⁴M for both GGF and naloxone. Taken together, these results indicatethat both the peptide, GGF, and the naloxone protected neurons fromLPS-induced DA neuron cell death and loss of function with a similarefficacy and dose response.

Example 12 Effects of Femtomolar Concentrations of GGF and Naloxone onthe Production of Various Species by Neuron-Glia Cultures

Primary enriched-microglia cultures were prepared as in Example 1. Thecultures were pretreated with varying concentrations of naloxone forabout 30 minutes and GGF followed by treatment with 10 ng/ml LPS as inExample 1. The intracellular ROS concentrations and superoxide amountswere measured as in Example 5. The data are expressed as the percent ofthe control cultures and are the mean ±SEM of three experimentsperformed in triplicate. *P<0.05, **P<0.01, compared to control.

Both 10⁻¹⁴M Nal and 10⁻¹⁴M GGF reduced intracellular ROS concentrationsby 65% (FIG. 12A) and reduced microglial superoxide response to nearlycontrol levels (FIG. 12B). These results demonstrate a similar efficacyand dose response of GGF and naloxone on microglial ROS levels, one ofthe pivotal signaling mechanisms governing microglia-mediatedneurotoxicity.

Example 13 Effect of Femtomolar Concentrations of GGF and Naloxone onMesencephalic Cultures from NADPH Oxidase Deficient Mice

The rat and mouse ventral mesencephalic neuron-glia cultures wereprepared as in Example 1. Mesencephalic neuron-glia cultures fromPHOX^(−/−) and PHOX^(+/+) mice were treated with either vehicle, LPS (5ng/ml), or were pretreated for 30 minutes with Naloxone or GGF (10⁻¹³M-10⁻¹⁴M) followed by addition of LPS (5 ng/ml) as in Example 1. DAneurotoxicity was measured by using the [³H] DA uptake assay as inExample 1. The data are expressed as the percent of the control culturesand are the mean ±SEM. The release of TNFα was measured with acommercially available enzyme-linked immunosorbent assay kit. *P<0.05,**P<0.01, compared to control. The amount of TNFα was measured as inExample 2.

Both naloxone and GGF failed to show neuroprotection in PHOX^(−/−)cultures (FIG. 13A), supporting that inhibition of this enzyme iscritical to the mechanism of action. The TNFα production was measured inresponse to LPS in PHOX^(−/−) and PHOX^(+/+) mesencephalic neuron/gliacultures pretreated for 30 minutes with GGF and Nal.

Again, PHOX^(−/−) mice failed to show any TNFα reduction in response toLPS with pretreatment of either neuroprotective compound, while thecontrol mice showed a reduction of TNFα with Nal and GGF treatment(10⁻¹⁴M) (FIG. 13B), demonstrating that these femtomolar actingcompounds also inhibit the ROS-induced amplification of TNFα expression.Together, these results support the conclusion that GGF and Nal affordneuroprotection through inactivation of NADPH oxidase.

The failure of Nal or GGF to protect against LPS-induced neurotoxicityin PHOX^(−/−) cultures indicates that NADPH oxidase may be a componentto the mechanism of neuroprotection.

Example 14 Molecular Modeling of Naloxone and GGF

Given the striking functional and mechanistic similarity betweennaloxone and GGF at femtomolar doses, it was thought these two compoundsmight act on the same site. To investigate this hypothesis, bothcompounds were compared for structural and chemical similarities. TheSearch Compare Module of the Accelrys Insight II software package wasused to provide a systematic conformational search of stericallypermitted conformations for both naloxone and the tripeptide,Gly-Gly-Phe. Accessible conformations of both molecules were thencompared and superimposed based on electrostatic potential similarity(as defined by the program Good, A. C., Hodgkin, E. E., Richards, W. G.,“Utilization of Gaussian Function for the Rapid Evaluation of MolecularSimilarity”, J. Chem. Inf. Comput. Sci, 32, 188-191, 1992) and stericshape similarity (as defined in the Search_Compare User Guide, Oct.1995, San Diego: Accelrys/Biosym/MSI, 1995) p. 2-3). The GGF peptide wasbuilt within the InsightII software suite using the Biopolymer builder.

A conformational search was defined based on rotatable bonds, where 166conformations were identified (66 of which were redundant) resulting in100 uniquely defined conformations. (The Phe ring was kept in one planarorientation and conformations rotating this ring were not explored).These conformations were energy minimized resulting in 42 distinctenergy-minimized conformations. These 42 GGF peptide conformations werecompared in terms of electrostatic and steric-shape similarity withnaloxone. Naloxone, as a fused ring compound, has less conformationalflexibility than the GGF tripeptide.

The steric similarity function for two low energy stable conformationsof naloxone and GGF was 0.854, (i.e. identical molecules would sharesteric similarity function=1.0; most dissimilar molecules, −1.0),indicating that the two molecules have the potential for exhibitingsimilar steric interactions and therefore could fit within a similarlyshaped binding pocket depicted in FIG. 14. This is particularlyintriguing and surprising because while both DynA (the full lengthsequence from which GGF is derived), and naloxone are known to bind thekappa opioid receptor, GGF is missing the first amino acid (tyrosine)required to bind the kappa receptor, suggesting that the similarity insteric conformations and interactions is critical to a site of actionindependent of the opiate receptors.

Example 15 Binding of Naloxone to NADPH

Binding affinities of naloxone for COS-7 cells transfected with gp91/p22was determined using either [³H]-(+)Naloxone or [³H]-(−)Naloxone (2 nM;PerkinElmer Life Sciences) as ligands and displaced with 10 μM of cold(−)Naloxone in HBBS containing 0.1% (W/V) fatty acids free albumin (lotB22558, Calbiochem). COS-7 cells transfected with gp91/p22 and COS-7cells that were not transfected (WT) were detached using Versene(1:5000, GibcoBRL, Life technologies). For intact cell assays, afterwashing twice with HBSS, cells were transferred to micro centrifugetubes at 10⁶ cells per tube.

To acquire membrane preparations, cells were lysed in buffer (20 nMTris, pH=7.4, 2 mM EDTA, 10 μg/ml CLAP) and homogenized. Lysate wastransferred to a 50 ml conical tube, where there were centrifuged at 250g for about 10 minutes at about 4 C. The supernatant was thentransferred to another tube and spun at 100,000 G for about 90 minutes.The supernatant was then discarded and the protein pellet wasre-suspended in 2 ml of lysis buffer. Finally, 50 μg of protein wasaliquoted into a 1.5 ml tube for further assay.

All competition reactions of either intact cells or membranepreparations were allowed to proceed at 4° C. Cells or membranepreparations were incubated with either [³H]-(+)Naloxone or[³H]-(−)Naloxone with gentle mixing in a roller drum for one hour.Experiments were terminated by rapid filtration through Glass fiberfilters (F4144-100EA, Sigma-Aldrich) using a sampling manifold(XX2702550, Millipore). After washing with HBSS four times, the filterswere collected and radioactivity retained on the filters was determinedby liquid Scintillation counting. All values are expressed aspercentages relative to the binding capacity of the wild type control.To determine whether COS-7^(gp91/p22) transfected cells had an increasedlevel of general non-specific binding, binding affinities of LPS forCOS-7 cells transfected with gp91/p22 was determined either with[³H]-naloxone (2 nM; PerkinElmer Life Sciences) as ligands displacedwith 10 μM of cold naloxonein HBBS containing 0.1% (W/V) fatty acidsfree albumin (lot B22558, Calbiochem).

The study showed that COS-7^(gp91/p22) cells, an immortal kidney cellline stably transfected with gp91/p22, have an increased bindingcapacity (150-180%) above the control COS-7 cells, which do not expressgp91 (NADPH oxidase membrane bound catalytic subunit) or p22 (NADPHoxidase membrane anchor protein) (FIG. 15). This data offers support forthe hypothesis that naloxone binds to the gp91 protein.

Example 16 In Vivo Effect of Dextromethorphan on TNFα(iROS, and alanineaminotransferase (ALT)

Animal studies were performed in accordance with National Institutes ofHealth Guidelines and with the approval of the Institute's Animal Careand Use Committee, and followed NIH guidelines. Male, CD-1 mice(6-week-old) were purchased from Charles River laboratories, fed on astandard diet and with tap water ad libitum for two weeks. Environmentalconditions were standardized, including a room temperature of 21° C. and12 hours artificial lighting. Mice were fasted 12 hrs before use.

Endotoxic shock was induced in the mice by administering a singleintraperitoneal dose of lipopolysaccharide/D-(+/−)Glactosomine (Sigma,St. Louis, Mo.) (LPS/GalN) (20 μg/700 mg/kg) in saline. To test whetherDM has protective effects on septic shock, varying concentrations (6.25,12.5 and 25 mg/kg) of DM were injected into the mice subcutaneously 30min before, and 2, and 4 hr after the injection of LPS/GalN. Controlmice received the same amount of saline. At different time points, theanimals were killed, and serum and liver samples were collected.

To examine the therapeutic effects of DM, animals were treated with 12.5mg/ml DM at 30 min before LPS/GalN injection, and 30, 60 and 120 minafter LPS/GalN administration. Serum ALT was measured to evaluate thetherapeutic effects of the DM. Survival rate was evaluated within 12hours after endotoxin administration.

Blood was collected from the eye while the mice were anesthetized, andthen perfused with saline. Perfused liver samples were collected andfrozen at −70° C. The blood samples were stored at 4° C. overnight andthen centrifuged at 1500×g at 4° C. for 15 min. Serum was collected andstored at −70° C. for ALT, and TNFα ELISA assays. The frozen liversamples were homogenized in 10 mg/ml cold lysis buffer (20 mM Tris, 0.25M sucrose, 2 mM EDTA, 10 mM EGTA, 1% Triton X-100 and protein cocktailinhibitor), and then centrifuged at 35,000×g for 40 min. The supernatantwas then collected for protein assay using BCA Protein Assay Reagent Kit(Prod# 23227, PIERCE), and ELISA for TNFα.

Serum alanine aminotransferase (ALT) activity was assayed as a marker ofhepatocellular death using a commercially available kit (Infinite ALT,Sigma, St. Louis, Mo.). A portion of the liver was fixed in 10% neutralformalin, processed by standard histological techniques, stained withhematoxylin and eosin, and examined for morphological evidence of liverinjury.

The levels of TNFα in the serum and liver were determined as in Example1.

Kupffer cell samples were collected by anesthetizing the CD-1 mice withpentobarbital anesthesia [60 mg/kg intraperitoneally (i.p.)]. Theabdomen of the animals was shaved and opened, the portal vein wascannulated and perfused with Ca²⁺- and Mg²⁺-free Hanks' balanced saltsolution (HBSS) at 37° C. for 5 min at a flow rate of 13 ml/min.Subsequently, the liver was perfused with HBSS containing 0.05%collagenase IV (Sigma Chemical, St. Louis, Mo.) at 37° C. for 5 min.After the liver was digested, it was excised and cut into small piecesin collagenase buffer. The suspension was filtered through nylon gauzemesh, and the filtrate was centrifuged at 450×g for 10 min at 4° C. Cellpellets were resuspended in buffer, parenchymal cells were removed bycentrifugation at 50×g for 3 min, and the nonparenchymal cell fractionwas washed twice with buffer. Cells were centrifuged on a densitycushion of 50% of Percoll (Pharmacia, Upsala, Sweden) at 1,000×g for 15min, and the Kupffer cell fraction was collected and washed again withbuffer. The viability of the cells was determined by tryptan blueexclusion at >90%. The cells were seeded onto 24-well culture plates andcultured in RPMI 1640 (GIBCO Laboratories Life Technologies, GrandIsland, N.Y.) supplemented with 10% fetal bovine serum and antibiotics(100 U/ml penicillin G and 100 μg/ml streptomycin sulfate) at 37° C.with 5% CO₂. Non-adherent cells were removed after 2 hours by replacingmedia, and cells were cultured for 24 hours before the experiments.

The production of superoxide and intracellular ROS by the Kupffer cellswere measured as in Example 5.

The survival rate of the mice at 12 hours post-LPS/GalN treatment arepresented in Table 2.

TABLE 2 Dextromethorphan Animal number Rate (mg/kg) (n) Survived animalof survival (%) 0 61 28 45.9 6.25 12 8 66.6 12.5 56 50 89.3 25 12 1191.6

About 44% percent of animals in LPS/GalN alone group died within 12hours of LPS/GalN challenge. Pretreatment with DM (25 and 12.5 mg/kg,i.c.) significantly increased the survival rate up to about 90% (11/12and 50/56 mice survived). Even at the lower concentration (6.25 mg/kg,i.c.), DM pretreatment increased survival rate to 67%. This clearlyshows that DM is effective in protecting LPS/GalN-induced lethal shockin mice. This effect was also observed with DM i.c. 30 min post LPS/GalNchallenge.

The liver samples collected from the LPS/GalN-induced mice are shown inFIGS. 16A (control), 16B (12 hours after LPS/GalN) and 16C (animaltreated with DM). The liver histology was examined to evaluate theeffect of DM treatment on LPS/GalN hepatotoxicity. In the LPS/GalN andsaline treated mice, the liver sections showed apparently broadhemorrhagic necrosis and apoptosis, and severe hepatocyte swelling 12hours after LPS/GalN challenge (FIG. 16B, arrows). These pathologicalalterations were dramatically ameliorated in the liver of animalsreceiving DM treatments (FIG. 16C). In the LPS/GalN plus DM-treatedanimals, hepatic congestion and hepatocellular necrosis were rareevents.

Serum alanine aminotransferase (ALT), an indicator of acutehepatocellular death, was examined on DM treated mice to determine theprotective effect of DM. Serum ALT increased ˜35-fold over controls by12 hrs after LPS/GalN administration (FIG. 17), DM dramaticallydecreased serum ALT level in a dose-dependent manner and reduced toabout 25% of LPS/GalN group at 12.5 and 25 mg/kg of DM. Time-dependentreduction of serum ALT level is shown in FIG. 18, DM administrated atdifferent time points, including 30 min pre-treatment and 30, 60, 120min post-treatments shows protective effects to various extracts, thelater the DM treatment, the less protective.

TNFα, an important factor that plays an important role in sepsis, wasstudied as a mechanism of the protective effect of DM inLPS/GalN-challenge mice. DM (12.5 mg/kg, i.c.) was administered to mice,followed by LPS/GalN challenge 30 min later. Serum and liver TNFα levelwas assessed using ELISA at the indicated time points. As shown in FIGS.19A and 19B, DM significantly decreased TNFα level in both serum andliver at 1.5 and 2 hours after LPS/GalN challenge. The suppression ofTNFα level was also found in DM 30 min post LPS/GalN treatment (data notshown). The reduction of hepatic TNFα paralleled the reduction of serumTNFα, indicating that decrease TNFα is an important mechanism ofprotection.

Activation of Kupffer cells by LPS is a critical event in theendotoxemia or sepsis. Therefore Kupffer cells were isolated to studythe possible mechanism of DM in protection liver injury and sepsis.Endotoxin activates Kupffer cells to release inflammatory mediators suchas free radicals and TNFα.

This study showed that both extracellular and intracellular superoxideproduction in CD-1 mouse Kupffer cells was increased significantly byLPS 10 ng/ml stimulation. This increase was significantly blunted by DMat dosage of 10 μM, and attenuated by 5 μM DM, as seen in FIGS. 20A and20B.

Example 17 Gene Expression Studies

Total RNA was extracted from liver tissues (n=4 to 5) by Trizol reagent(Sigma, St. Louis, Mo.) and purified with an RNeasy column (Qiagen,Valencia, Calif.). Expression of the selected genes was quantified usingreal-time RT-PCR analysis that began by reverse transcribing the sampleswith MuLV reverse transcriptase and oligo-dT primers. The forward andreverse primers for the selected genes were designed using PrimerExpress software and are listed in Table 3.

TABLE 3 Accession Forward Reverse Gene Number Primer Primer MIP-2NM_009140 CCTCAACGGAA CTCAGACAGCG GAACCAAAGAG AGGCACATC (Seq. ID (Seq.ID No. 3) No. 4) TSP1 M87276 GCCGGATGACA GCCTCAAGGAA AGTTCCAA GCCAAGAAGA(Seq. ID (Seq. ID No. 5) No. 6) mKC NM_008176 TGGCTGGGATT GTGGCTATGACCACCTCAAG TTCGGTTTGG (Seq. ID (Seq. ID No. 7) No. 8) ICAM-1 NM_010493GTCTCGGAAGG CGACGCCGCTC GAGCCAAGTA AGAAGAA (Seq. ID (Seq. ID No. 9) No.10) IL-6 J03783 GCCCACCAAGA GAAGGCAACTG ACGATAGTCA GATGGAAGTCT (Seq. ID(Seq. ID No. 11) No. 12) IL-10 M37897 CCAAGCCTTAT GATTTCTGGGCCGGAAATGATC CATGCTTCTC (Seq. ID (Seq. ID No. 13) No. 14) c-jun/AP-1J04115 ACTCCGAGCTG CCCACTGTTAA GCATCCA CGTGGTTCATG (Seq. ID (Seq. ID No.15) No. 16) c-myc X01023 CGCCGCTGGGA TCCTGGCTCGC AACTTT AGATTGTAA (Seq.ID (Seq. ID No. 17) No. 18) GADD45 L28177 CAGATCCATTT TCCAGTAGCAGCACCCTCATCC CAGCTCAGC (Seq. ID (Seq. ID No. 19) No. 20) GADD153 X67083CTCCTGTCTGT TACCCTCAGTC CTCTCCGGAA CCCTCCTCA (Seq. ID (Seq. ID No. 21)No. 22) beta-actin M12481 GTATGACTCCA GGTCTCGCTCC CTCACGGCAAA TGGAAGATG(Seq. ID (Seq. ID No. 23) No. 24)Abbreviation are: MIP-2, Macrophage Inflammatory Protein-2; TSP-1,thrombospondin1; mKC, a mouse CXC chemokine; ICAM-1, Intercellular CellAdhesion Molecule-1; IL-6, Interleukin-6; IL-10, Interleukin-10;GADD153, Growth Arrest and DNA Damage inducible protein 153; GADD45,Growth Arrest and DNA Damage inducible protein 45.

The SYBR green DNA PCR kit (Applied Biosystems, Foster City, Calif.) wasused for real-time PCR analysis. The relative differences in expressionbetween groups were expressed using cycle time (Ct) values and therelative differences between groups were expressed as relative increasessetting control as 100%. Assuming that the Ct value is reflective of theinitial starting copy and that there is 100% efficacy, a difference ofone cycle is equivalent to a two-fold difference in starting copy.

The results of the gene expression studies can be seen in Table 4 below.

TABLE 4 LPS/GalN Control alone LPS/Cont LPS + DM LPS + DM/ContInflammatory Markers MIP-2 1.0 ± 0.3 153.3 ± 6.4  153.3 39.0 ± 2.1* 39.0Thrombospondin-1 1.0 ± 0.1 94.2 ± 2.5  94.2 56.0 ± 1.57* 56.0 mKC 1.0 ±0.2 13.7 ± 2.9  13.7  5.8 ± 1.6* 5.8 ICAM-1 1.0 ± 0.1 8.45 ± 1.3  8.455.05 ± 2.7* 5.05 IL-6 1.0 ± 0.2 153.4 ± 13.3  153.4 43.6 ± 6.3* 43.6IL-10 1.0 ± 0.1 25.3 ± 2.9  25.3 15.0 ± 3.3* 15 AcutePhase Protein genes& Cell-Death Markers c-jun/AP-1 1.0 ± 0.1 28.0 ± 7.5  28.0  9.1 ± 0.8*9.1 c-myc 1.0 ± 0.07 45.4 ± 3.6  45.4 18.4 ± 2.1* 18.4 GADD45 1.0 ± 0.224.6 ± 12.4 24.6  5.3 ± 2.4* 5.3 GADD153 1.0 ± 0.1 7.4 ± 0.3 7.4  2.7 ±1.3* 2.7 Mice were given GalN/LPS (700 mg/20 μg/kg, ip), or GalN/LPS +DM (12.5 mg/kg, sc, x2). Liver samples were taken at 12 hr afterGalN/LPS administration, and total RNA was isolated for real-time RT-PCRanalysis. In each individual sample, the expression level of each genewas first normalized with that of β-actin and then the relativedifferences between groups were expressed as relative increases settingcontrols as 1.0.Data represent means ± SE of n = 4-5 animals per group.*P < 0.05 (compared to GalN/LPS alone.) Gene abbreviations are listed inTable 3.

As shown in Table 4, 12 hrs after GalN/LPS, there were dramaticincreases in the expression of mouse macrophage inflammatory protein(MIP-2, 153.3-fold), thrombospondin-1 (TSP1, 94.2-fold), mouse chemokine(mKC, 13.7-fold), intracellular adhesion molecule-1 (ICAM-1, 8.45-fold),interleukin-6 (IL-6, 153.4-fold), and interleukin-10 (IL-10, 25.3-fold)genes. DM significantly diminished the GalN/LPS-induced enhancedexpression for the MIP-2, TSP1, mKC, ICAM-1, IL-6 and IL-10 genes.

GalN/LPS acute hepatotoxicity also greatly enhanced the expression ofc-jun/AP-1 (28-fold), c-myc (45.4-fold), while the expression of bothgenes was diminished to 9.1- and 18.4-fold, respectively with DMtreatment. As a result of GalN/LPS toxicity, the DNA damage responsibleproteins such as GADD45 and GADD153 were also increased by 24.6 and7.4-fold respectively. There was a significant suppression ofGalN/LPS-induced GADD45 and GADD153 protein genes by DM to 5.3 and2.7-fold respectively.

Example 18 In Vitro Effects of DM on Septic Shock

Endotoxic shock were induced in mice in the same was as Example 16. Themice were given LPS/GalN (20 μg/700 mg/kg, ip) with or without theadministration of DM (10 mg/kg, 1 μg/kg and 100 pg/kg, s.c.) 30 minbefore LPS/GalN. The survival rate of the animals was evaluated 12 hrsafter LPS/GalN treatment. The results are shown in Table 5 below and aredisplayed in FIG. 21.

TABLE 5 Amount of dextromethorphan Number of animals No. of animals Rateadministered in group survived of survival % 0 30 17 56.7 10 mg/kg 26 2284.6 1 ug/kg 17 10 58.8 100 pg/kg 47 36 76.6

FIG. 22 shows the levels of serum ALT, measured as in Example 16.

Sections of the mice livers were stained with hematoxylin and eosin, andphotomicrographs were taken at 100× magnification. FIG. 23A shows aphotomicrograph of a liver sample with LPS/GalN alone. As seen there,there is a foci of necrotic parenchymal cells, cell swelling, andcongestion. FIG. 23B shows a photomicrograph of a liver sample that wasadministered 10 mg/kg DM plus LPS/GalN. As seen there, hepaticcongestion and cell death are mild, while the cell swelling is the onlynotable lesion. FIG. 23C shows a photomicrograph of a liver sample withthat was administered 1 μg/kg DM plus LPS/GalN. As seen there, hepaticcongestion and cell death are obvious. FIG. 23D shows a photomicrographof a liver sample that was administered 100 pg/kg DM plus LPS/GalN. Asseen there, cell swelling is the only notable lesion, but cell death ismild.

Kupffer cells were pre-treated with DM (10⁻⁵, 10⁻¹⁰ or 10⁻¹⁴ M) orvehicle (control or LPS treated group) for 30 min, and then stimulatedwith LPS 5 ng/ml or vehicle (control group). TNF-α production (6 hrsafter LPS stimulation) was measured by an ELISA kit as in Example 5. Thedata, which are seen in FIG. 24 are the mean ±SEM of 3-4 individualexperiments with triplicates. *, Significantly different from LPS alonetreated culture, P<0.05.

Action of VPA in Model of Inflammation-Mediated DopaminergicNeurodegeneration

Examples 19 through 22 show VPA protects dopaminergic neurons fromLPS-induced neurotoxicity through the inhibition of microglialactivation. The anti-inflammatory responses of cultures stimulated withLPS and pretreated with VPA are also characterized.

Statistical Analysis: The data were presented as the mean ±S.E.M. Formultiple comparisons of groups, ANOVA was used. Statistical significancebetween groups was assessed by paired or unpaired Student's t-test, withBonferroni's correction. A value of p<0.05 was considered statisticallysignificant.

Example 19 Effect of VPA on LPS-Induced Degeneration of DopaminergicNeurons

The effect of concentration-dependent VPA pretreatment on LPS-inducedneurotoxicity in dopaminergic neurons in rat primary mesencephalicneuron-glia cultures is shown in FIG. 25. Neuron-glia cultures wereprepared from the ventral mesencephalic tissues of embryonic day 13-14rats. Dissociated cells were seeded at 1×10⁵/well and 5×10⁵/well topoly-D-lysine-coated 96-well and 24-well plates, respectively. Cellswere maintained at 37° C. in a humidified atmosphere of 5% CO₂ and 95%air, in minimal essential medium (MEM) containing 10% fetal bovineserum, 10% horse serum, 1 gm/l glucose, 2 mM L-glutamine, 1 mM sodiumpyruvate, 100 μM non-essential amino acids, 50 U/ml penicillin, and 50μg/ml streptomycin. At the time of treatment of 6-day old cultures, themedium was changed to 2% MEM (2% fetal bovine and horse serum in MEM)and the cultures were then pretreated with vehicle or the indicatedconcentration (0.05, 0.2, 0.4 and 0.6 mM) of VPA (Sigma-Aldrich, St.Louis, Mo., which was freshly prepared with culture medium. Two dayslater, cells were treated with 20 ng/ml LPS 3 days and followed by theassay of [³H]DA uptake. [3H]DA uptake assays were performed as describedabove in Example 1.

Degeneration of dopaminergic neurons was assessed by measuring theability of cultures to take up [3H]DA, or counting the number of TH-irneurons after immunostaining (see below). The [³H]DA uptake assay showedthat LPS treatment reduced the capacity of the cultures to take up DA toapproximately 50% of the vehicle control and this LPS-induced reductionwas concentration-dependently prevented by VPA pretreatment (FIG. 25).At 0.6 mM VPA, which is within the therapeutic range of this drug, theLPS-induced decrease in DA uptake was completely restored and VPA aloneat this concentration did not affect DA uptake levels in the cultures.

The effect of VPA pretreatment on morphological changes of mesencephalicdopaminergic neurons immunostained with anti-TH antibody weredetermined. The primary midbrain cultures were pretreated with theindicated concentrations of VPA for 48 h and then treated with 20 ng/mlLPS for 72 h, as described above. DA neurons were recognized with theanti-TH antibody and microglia were detected with the OX-42 antibody,which recognizes the CR3 receptor. Briefly, formaldehyde (3.7%)-fixedcultures were treated with 1% hydrogen peroxide (10 ml) followed bysequential incubation with blocking solution (30 min), primary antibody(overnight, 4° C.), biotinylated secondary antibody (2 h), and ABCreagents (40 min). Color was developed with 3,3′-diaminobenzidine. Formorphological analysis, the images were recorded with an invertedmicroscope (Nikon, Tokyo, Japan) connected to a charge-coupled devicecamera (DAGE-MTI, Michigan City, Ind.) operated with MetaMorph software(Universal Imaging Corporation, Downingtown, Pa.). TH-ir neurons in eachwell of the 24-well plate were visually counted under the microscope at400× magnification. The results are shown in FIG. 26. Images wererecorded with an inverted microscope connected to a charge-coupleddevice camera. Scale bar, 25 μm.

Morphological inspection revealed that LPS treatment not only decreasedthe number of TH-ir neurons, but also caused a loss of neuronal process(FIGS. 27A and 27B). These characteristics were reversed by VPApretreatment in a dose-dependent manner (FIG. 27D-27F). LPS-induced lossof TH-ir neurons was prevented by VPA pretreatment in aconcentration-dependent manner with a significant effect at 0.2, 0.4 and0.6 mM. VPA at 0.6 mM, either alone or in conjunction with LPS, enhancedthe TH immunostaining in both the cell bodies and neuronal processescompared with the vehicle control (FIGS. 27C and 27F).

Data are expressed as means ±S.E.M. from 4 independent experiments. *,p<0.05 compared with LPS-treated cultures; †, p<0.05, ‡, p<0.01,compared with untreated control.

Example 20 VPA Pretreatment Suppresses LPS-Induced Activation ofMicroglia And Production of Pro-Inflammatory Factors in Neuron-GliaCultures

FIGS. 28A-28F show VPA pretreatment suppresses LPS-induced microgliaactivation revealed by OX-42 immunostaining. Mesencephalic neuron-gliacultures were pretreated with VPA for 48 h and then treated with 20ng/ml LPS for 72 h, as described in Example 19. Immunostaining with anantibody against OX-42 was then performed, as described in Example 19.

Mesencephalic neuron-glia cultures treated with LPS displayed thecharacteristics of activated microglia such as, increased cell size,irregular shape, and intensified OX-42 immunoreactivity, a specificmarker for rat microglia as shown in FIGS. 28A and 28B. TheLPS-stimulated activation of microglia was suppressed in neuron-gliacultures pretreated for 48 h with 0.4 or 0.6 mM VPA, as shown in FIGS.28D through 28F. VPA alone did not show significant effects on microgliaactivation, as shown in FIG. 28C. Scale bar, 100 μm. The images shownare representative of 3 independent experiments.

Suppression of LPS-induced release of pro-inflammatory factors from ratprimary midbrain cultures by VPA pretreatment is shown in FIGS. 29 and30. Activation of microglia mediates the LPS-induced dopaminergicneurodegeneration. This process has been attributed, at least in part,to secretion of a variety of pro-inflammatory and neurotoxic factors,such as TNFα, NO, and superoxide.

Mesencephalic neuron-glia cultures were pretreated with VPA or vehiclecontrol for 48 hours prior to stimulation with 20 ng/ml LPS. The releaseof TNF-αwas determined 3 hours after LPS treatment. The release of TNFαwas measured with a rat TNFα enzyme-linked immunosorbent assay kit fromR & D System (Minneapolis, Minn.). As shown in FIG. 29, pretreatmentwith 0.4 or 0.6 mM VPA completely blocked LPS-induced production of TNFαin neuron-glia cultures determined at 3 h after LPS stimulation. Even at0.2 mM VPA, LPS-induced TNFα production was also significant inhibited.

Production of nitric oxide (NO) was determined in the mesencephalicneuron-glia cultures, following pretreatment and LPS stimulation asdescribed above, by measuring the accumulated levels of nitrite in thesupernatant with Griess reagent. Accumulation of nitrite, an indicatorof LPS-stimulated production of NO, was determined 24 after LPSstimulation. As shown in FIG. 30, pretreatment with 0.4 and 0.6 mM VPAreduced LPS-stimulated NO production 54% and 78% of the control,respectively.

Results are means ±S.E.M of 4 independent experiments. *, p<0.05compared with LPS-treated cultures; †, p<0.05, compared with untreatedcontrol.

Example 21 VPA Pretreatment Inhibits LPS-Induced Intracellular ReactiveOxygen Species Production in Enriched Microglia

To determine if VPA pretreatment protects dopaminergic neurons againstintracellular oxidative stress, the level of intracellular reactiveoxygen species (iROS) was measured via DCF oxidation in enrichedmicroglia cultures.

Assay of intracellular ROS is performed as follows.5-(and-6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate(CM-H2-DCFDA) (Molecular Probes, Eugene, Oreg.), a chloromethylderivative of H2-DCFDA, passively diffuses into cells in which it ishydrolyzed by intracellular esterases to liberate2′-7′-dichlorofluorescein (DCF) which, during reaction with oxidizingspecies, yields a highly fluorescent compound that is trapped inside thecell. Microglia-enriched cultures were prepared from the whole brains of1-day-old rats as described in Example 1. Immunocytochemical analysisindicated that the cultures were 95-98% pure for microglia. Cells wereseeded at 1×10⁵/well in 96-well plates for one day followed by treatmentwith 0.6 mM or 1.2 mM VPA for 24 h. The cultures were used for the assayof intracellular ROS.

After washing two times with warm Hank's balanced salt solution (HBSS),CM-H2-DCFDA, diluted to a final concentration of 1 μM in phenol red-freeHBSS, was added to cultures and incubated for 30 min at 37° C. Thencultures were added with 0.6 or 1.2 mM VPA in HBSS again for 30 min andfollowed the treatment with 100 ng/ml LPS for 2 hours at 37° C.,fluorescence intensity was measured at 485 nm for excitation and 530 nmfor emission using a SpectraMax Gemini XS fluorescence microplate reader(Molecular Devices, Sunnyvale, Calif.).

The iROS level was significantly increased by LPS treatment and thisincrease was completely blocked by pretreatment with VPA at 0.6 or 1.2mM (FIG. 31). VPA alone at 0.6 mM, but not 1.2 mM, also reduced basaliROS levels.

Example 22 VPA Treatment Decreases the Number of Microglia

Primary rat microglia-enriched cultures were used to determine if VPAtreatment affected the total number of microglia. Microglia cell numberwas determined as follows. Primary microglia-enriched cultures wereprepared from the whole brains of 1-day-old rats as describedpreviously. After one day in vitro were treated with the indicatedconcentrations of VPA (0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, or 1.2 mM)for 48 h, or with 0.6 mM VPA for different times (6, 12, 24 or 48 h).After treatment with VPA and vehicle for 48 hours, the number andmorphology of microglia in cultures were observed under an invertedmicroscope (Nikon, Tokyo, Japan) at 100×. The total number of microgliawas counted by using the CyQUANT cell proliferation assay kit (MolecularProbes, Inc.).

Microscopic examination showed that 0.6 mM VPA time-dependentlydecreased the number of microglia with a significant effect at 24 h and48 h after treatment (FIGS. 32A-32F and 33). The VPA effect was alsoconcentration-dependent with a robust decrease in microglia number inthe dose range of 0.2 mM to 1.2 mM after 48 h treatment (FIGS. 34A-34Dand 35). The loss of microglia was about 80% by treatment with 0.8 mMVPA. Moreover, VPA-induced microglia loss was time and dose-dependentlyassociated with aggregations or clumping of surviving cells (FIGS.32A-32F and 34).

Example 23 Survival-Promoting Effects of VPA Against Spontaneous DANeuronal Death

VPA dose-dependently induces survival-promoting effects againstspontaneous DA neuronal death in rat primary mesencephalic neuron-gliacultures.

In Example 23, [³H]DA uptake assay and immunohistochemical analysis forTH-IR neurons were used to assess the viability of DA neurons in ratprimary mesencephalic neuron-glia cultures in which approximately 1% ofthe neurons are dopaminergic. [³H]DA uptake assays were performed asdescribed in Example 1.

Rat primary mesencephalic neuron-glia cultures were prepared from theventral mesencephalic tissues of embryonic day 13-14 Fisher 344 rats.Dissociated cells were seeded at a density of 5×10⁵/well topoly-D-lysine-precoated 24-well plates. Cells were maintained at 37° C.in a humidified atmosphere of 5% CO₂ and 95% air, in minimal essentialmedium containing 10% fetal bovine serum (FBS), 10% horse serum, 1 gm/lglucose, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM non-essentialamino acids, 50 U/ml penicillin, and 50 μg/ml streptomycin. Unlessotherwise indicated, seven-day-old cultures were used for treatment. Atthis time, immunocytochemical analysis indicated that the ratneuron-glia cultures contained 11% microglia, 48% astroglia, and 41%neurons, among which about 1% of cells represent tyrosinehydroxylase-immunoractive (TH-IR) neurons.

Valproic acid (VPA) (Sigma-Aldrich, St. Louis, Mo.) is prepared as asolution in double-distilled water and then sterile filteringimmediately before use.

Seven days after seeding, each well was treated with indicatedconcentration of VPA or its vehicle. Seven days after treatment, theviability of dopaminergic neurons was assessed by [³H]DA uptake assays.Quantified results are expressed as mean ±SEM of percentage ofvehicle-treated cultures from three experiments performed in duplicate.*, p<0.05 compared with the corresponding vehicle-treated controlcultures.

As shown in FIG. 36, results indicated that VPA inducedsurvival-promoting effects in a dose-dependent manner. At 0.6 mM, VPAsignificantly protected DA neurons from spontaneous neuronal death.

Next, the rat primary mesencephalic neuron-glia cultures were treatedwith 0.6 mM VPA for various times to determine the treatmenttime-dependency. Rat primary mesencephalic neuron-glia cultures in a24-well plate were treated with 0.6 mM VPA or its vehicle for indicatedtime 7 days after seeding. The viability of dopaminergic neurons wasassessed by [H]DA uptake assays, shown in FIG. 37 or counting of TH-IRneurons, shown in FIG. 38. Quantified results are expressed as mean ±SEMof percentage of vehicle-treated control cultures from three experimentsperformed in duplicate. *, p<0.05 compared with the correspondingvehicle-treated control cultures. VPA treatment increased the capacityof [³H]DA uptake and the number of TH-IR neurons in a time-dependentmanner, as shown in FIGS. 37 and 38. In both cases, treatment with 0.6mM VPA for 7 days, but not 3 or 5 days, resulted in a marked increase inthe parameter compared with vehicle-treated control. VPAtime-dependently induces survival-promoting effects against spontaneousDA neuronal death in rat primary mesencephalic neuron-glia cultures.

Example 24 Roles of Astroglia in VPA-Induced Neurotrophic Effects

Whether astroglial cells are involved in the neurotrophic actions of VPAwas tested by preparing media conditioned by incubation of astroglialcultures in the absence or presence 0.6 mM VPA. The results are shown inFIG. 39.

Mixed-glia cultures were first prepared from brains of 1-day-old Fisher344 rat pups, as described previously (Liu, et al. 2001). Mechanicallydissociated brain cells (5×10⁷) were seeded onto 150 cm² culture flasksin Dulbecco's modified Eagle's medium containing 10% heat-inactivatedFBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μM non-essential aminoacids, 50 U/ml penicillin, and 50 μg/ml streptomycin. The cultures weremaintained at 37° C. in a humidified atmosphere of 5% CO₂ and 95% air,and medium was replenished 4 days after the initial seeding. Uponreaching confluence (usually 12-14 days later), microglia were detachedfrom astroglia by shaking the flasks for 5 hours at 180 rpm. Astrogliawere then detached with trypsin-EDTA and seeded in the same culturemedium. After five or more consecutive passages, cells were seeded onto24-well plates (10⁵/well) for experiments. Immunocytochemical stainingof the astroglia enriched cultures with either anti-GFAP or anti-OX-42antibody indicated astroglial purity of greater than 98% and less than2% of microglia contamination.

To exclude the possibility that the effects of ACM-VPA were due to thepresence of VPA in the conditioned medium, ACM-VPA was dialyzedovernight using the Slide-A-Lyzer® Dialysis Cassette (PierreBiotechnology, Inc) to remove small molecular weight substances. Thedialyzed ACM-VPA still retained its ability to enhance DA uptake (datanot shown here).

The astrocyte-conditioned medium (ACM) derived from incubation withastrocytes for 12, 24 or 48 hours showed an approximate 4-fold increasein DA uptake following incubation of mesencephalic neuron-enrichedcultures with ACM for 7 days. The conditioned medium derived fromincubation of astrocytes in the presence of 0.6 mM VPA (ACM-VPA)displayed a more robust (more than 8-fold), time-dependent increase inDA uptake, compared with ACM. Exposure of the neuron-enriched culturesto 0.6 mM VPA for 7 days caused a less than 2-fold increase in DAuptake, suggesting that a direct action on neurons does not play a majorrole in VPA-induced neurotrophic effects.

Conditioned medium derived from rat primary astroglial cultures treatedwith vehicle (ACM) or 0.6 mM VPA (ACM-VPA) were harvested after 12, 24and 48 hours of incubation. Midbrain neuron-enriched cultures seeded in24-well plates at a density of 5×10⁵ cells/well were treated withvehicle, VPA, ACM or ACM-VPA for 7 days. Neurotrophic effect wasquantified by [³H]DA uptake assay.

The data are expressed as mean ±SEM of percentage of vehicle controlfrom four to five independent experiments performed in triplicate; *,p<0.001 compared with the corresponding vehicle control cultures; †,p<0.001 compared with the corresponding ACM-treated cultures.

Neuron-enriched cultures were immunostained with MAP-2 for morphologicalexamination. FIGS. 40A-40D show morphological features ofneuron-enriched cultures were examined after incubation with vehicle(A), 0.6 mM VPA (B), ACM (C) or ACM-VPA (D) for 7 days and thenimmunocytostaining with MAP-2 antibody.

The immunostaining was carried out according to the following procedure.Formaldehyde (3.7%)-fixed cultures were treated with 1% hydrogenperoxide (10 min) followed by sequential incubation with blockingsolution (30 min), primary antibody (overnight, 4° C.), biotinylatedsecondary antibody (2 hours), and ABC reagents (40 min) (VectorLaboratories, Burlingame, Calif.). The color development was achieved bythe addition of 3,3′-diaminobenzidine. For morphological analysis, theimages were recorded with an inverted microscope (Nikon, Tokyo, Japan)connected to a charge-coupled device camera (DAGE-MTI, Michigan City,Ind.) operated with the MetaMorph software (Universal ImagingCorporation, Downingtown, Pa.). For visual counting of TH-IR neurons,nine representative areas per well of the 24-well plate were countedunder the microscope at 100× magnification.

The results of the morphological examination are shown in FIGS. 20A-40D.Morphological examination of neuron-enriched cultures immunostained withMAP-2 demonstrated a dramatic increase in neurite outgrowth followingexposure for 7 days to ACM-VPA conditioned with 48 hours incubation withastrocytes. (FIG. 40D), compared with the vehicle-treated controlcultures (FIG. 40A). Much smaller effects were observed followingincubation with the VPA alone or corresponding ACM (FIGS. 40B and 40C).

To specifically visualize morphological changes in DA neurons, weimmunostained neuron-enriched cultures with anti-TH antibody. ACM andACM-VPA were harvested after 48 hours incubation of astrocyte withvehicle and 0.6 mM VPA, respectively. Images shown are representative ofat least three independent experiments. FIG. 30 shows morphologicalfeatures of neuron-enriched cultures were examined after incubation withACM (A) or ACM-VPA (B) for 7 days and then immunocytostaining with TH-IRantibody. Results demonstrated higher density of DA neurons, morecomplex neurite configurations and more neuronal connections inACM-VPA-treated cultures (FIG. 41A) than ACM-treated cultures (FIG.41B).

Example 25 GDNF as a Mediator of VPA-Induced Astroglia-DerivedNeurotrophic Effects

To test that GDNF is VPA-induced, astroglia-secreted neurotrophicsubstance, we used real time PCR and ELISA to quantify GDNF mRNA andprotein levels, respectively.

Rat primary astroglias were exposed to 0.6 mM VPA for various timesranging from 6 to 48 hours. Total RNA was extracted from cells by Trireagent (Sigma) and purified with RNeasy columns (Qiagen, Valencia,Calif.). Expression of the selected genes was quantified using real-timeRT-PCR analysis. Briefly, total RNA was reverse transcribed with MuLVreverse transcriptase and oligo-dT primers. The forward and reverseprimers for selected genes were designed using Primer Express software.The SYBR green DNA PCR kit (Applied Biosystems, Foster City, Calif.) wasused for real-time PCR analysis. The relative differences in expressionbetween groups were expressed using cycle time (Ct) values and therelative differences between groups were expressed as relative increasessetting the control as 100%. Assuming that the Ct value is reflective ofthe initial starting copy and that there is 100% efficacy, a differenceof one cycle is equivalent to a two-fold difference in the startingcopy.

Quantified results, shown in FIG. 42, are expressed as mean ±SEM ofpercentage of vehicle-treated control cultures from four experimentsperformed in triplicate. Results showed that VPA treatment caused atime-dependent increase in GDNF mRNA levels in astroglial cultures. Thisincrease was about 180% at 12 hours, 265% at 24 hours and back to thecontrol value at 48 hours (FIG. 42).

Next, we used ACM-VPA to analyze secreted GDNF protein. ACM-VPA wasprepared according to Example 20 and collected 48 hours after incubationwith astroglia. In FIG. 43, ACM-VPA was analyzed for secreted GDNF byELISA. GDNF levels were measured with an ELISA kit (GDNF EmaxImmunoAssay System; Promega, Madison, Wis.), according to the protocolof the supplier. The levels of GDNF were expressed as pg per ml ofsupernatant. The assay sensitivity ranged from 16 to 1000 pg/ml. Resultsare expressed as pg/ml from three experiments performed in duplicate.ACM-VPA showed a 2.1-fold over the vehicle control in levels of GDNFprotein (39 vs 83 pg/ml) (FIG. 43).

To investigate whether GDNF-neutralization interfered with theVPA-induced effects on DA neurons, ACM-VPA was pretreated with goatanti-GDNF IgG overnight prior to the addition to mesencephalicneuron-enriched cultures.

Rat mesencephalic neuron-enriched cultures are prepared from dissociatedventral mesencephalic cells from embryonic day 13-14 Fisher 344 ratswere seeded first at a density of 5×10⁵/well to poly-D-lysine-precoated24-well culture plates. Twenty hours after plating,cytosine-β-D-arabinofuranoside (10 μM) was added to the cultures tosuppress the proliferation of non-neuronal cells, notably glia. Threedays later, the culture medium was replaced with the maintenance medium.Routinely, the seven-day-old neuron-enriched cultures were used fortreatment. At this time the neuron-enriched cultures contained less than0.1% microglia, and 8% astroglia, as revealed by immunochemicalanalysis. Of the Neu-N immunoractive neurons, 2.7-3.9% was TH-IRneurons.

Neutralization of GDNF was performed by the addition of 2 μg/ml goattotal anti-GDNF IgG (1:100 dilutions; R&D Systems, Minneapolis, Minn.)to ACM-VPA. The ACM-VPA was incubated overnight with 2 μg/ml of eithercontrol goat IgG or goat anti-GDNF IgG, and then added to themesencephalic neuron-enriched cultures for 7 days prior to measuring DAuptake capacity. [³H]DA uptake assays were performed according to theprocedure presented in Example 1. Results, shown in FIG. 44, areexpressed as means ±SEM of percentage DA uptake in the cultures treatedwith ACM-VPA from three independent experiments. *, p<0.05 compared withthe ACM-VPA (C) treated cultures. The GDNF-neutralizing antibodysignificantly reduced the ACM-VPA-induced increase in DA uptake capacityfollowing 7 days of incubation, while pretreatment with control IgG waswithout effect (FIG. 44).

All statistical analyses were performed with SPSS software v. 10.0, andp-values of ≦0.05 were considered significant in all tests. GDNFtranscript abundance was expressed as a ratio of actin internal control.All dose-response experiments were analyzed by one-way analysis ofvariance (ANOVA), with treatments as the independent variable, followedby Dunnett's test comparing each treatment to the vehicle.

Example 26 VPA Robustly Protects DA Neurons from Neurotoxicity Inducedby LPS and MPP⁺

Whether VPA also protects DA neurons against LPS-induced neurotoxicitywas investigated in the primary mesencephalic neuron-glia cultures.

Mixed-glia cultures were first prepared from brains of 1-day-old Fisher344 rat pups, as described previously (Liu, Wang et al. 2001). Briefly,mechanically dissociated brain cells (5×10⁷) were seeded onto 150-cm²culture flasks in Dulbecco's modified Eagle's medium containing 10%heat-inactivated FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 μMnon-essential amino acids, 50 U/ml penicillin, and 50 μg/mlstreptomycin. The cultures were maintained at 37° C. in a humidifiedatmosphere of 5% CO₂ and 95% air, and medium was replenished 4 daysafter the initial seeding. Upon reaching confluence (usually 12-14 dayslater), microglia were detached from astroglia by shaking the flasks for5 h at 180 rpm. Astroglias were then detached with trypsin-EDTA andseeded in the same culture medium. After five or more consecutivepassages, cells were seeded onto 24-well plates (10⁵/well) forexperiments. Immunocytochemical staining of the astroglia enrichedcultures with either anti-GFAP or anti-OX-42 antibody indicated aastroglial purity of greater than 98% and less than 2% of microgliacontamination.

Mixed-glia cultures were pretreated with various doses of VPA for 48hours and then expose to 10 ng/ml LPS for 5 days. Dose-dependent effectof VPA on LPS-induced DA neuron degeneration is shown in FIG. 45. LPStreatment reduced the uptake capacity of DA by 45% and this loss wasrobustly blocked by VPA pretreatment in a dose-dependent manner. Infact, at 0.6 mM, the DA uptake levels in the VPA-pretreated cultures,either alone or in conjunction with LPS, were significantly higher thanthose in the untreated control.

Time-dependent neuroprotective effects of VPA on LPS-induced DA neurondegeneration are shown in FIG. 46. The mixed-glia cultures seeded in a24-well plate were treated for indicated time with 0.6 mM VPA followedby treatment with 10 ng/ml LPS. While pretreatment with 0.6 mM for 6hours failed to induce a significant effect, a 12 hours-pretreatmentproduced a complete neuroprotection against LPS neurotoxicity and a 48hours-pretreatment caused a further increase in DA uptake levels (FIG.46).

Morphological assessments of dopaminergic neurons in the primarymesencephalic neuron-glia cultures are shown in FIGS. 47A-47F. Thecultures were treated with vehicle alone (A), 0.6 mM VPA alone (B), 10ng/ml LPS alone (C) or pretreated for 48 hours with 0.2 (D), 0.4 (E) or0.6 mM VPA (F) followed by treatment with 10 ng/ml LPS. Seven dayslater, cultures were immunostained with anti-TH antibody. Images shownare representative of three separate experiments.

Morphological assessments of DA neurons immunostained with anti-THantibody revealed that VPA treatment promoted neurite formation andinter-neuronal networks (FIGS. 47A and 47B). In contrast, LPS treatmentcaused degeneration of DA neuronal soma and loss of neuritis anddendritic nodes (FIG. 47C). These LPS-induced morphological changes weredose-dependently, prevented by VPA pretreatment in the concentrationrange examined (0.2 to 0.6 mM).

Finally, we investigated whether VPA is able to protect againstneurotoxicity induced by MPP⁺, another PD-inducing toxin, in themesencephalic neuron-enriched cultures, which contains a much reduced %of astroglia (8% in the neuron-enriched culture vs. 50% in theneuron-glia culture). The MPP⁺ model in neuron-enriched cultures couldprovide a clue to determine the directly protective effect of VPA onneurons, since it is known that MPP⁺ exerted direct DA neurotoxicity.Neuron-enriched cultures were prepared according the description inExample 25.

Neuroprotective effects of VPA on MPP⁺ induced DA neurodegeneration inthe mesencephalic neuron-enriched cultures is shown in FIG. 48.Neuron-enriched cultures seeded in 24-well culture plates werepretreated for 48 hours with 0.6 mM VPA followed by treatment with 0.5μM MPP⁺. [³H]DA uptake was measured 7 days after MPP⁺ treatmentaccording to procedures described in Example 1. Results are expressed asmean ±SEM of percentage of vehicle control from three independentexperiments performed in duplicate. *, p<0.05 compared withvehicle-treated cultures. †, p<0.05 compared with correspondingMPP⁺-treated cultures.

Treatment with 0.5 μM MPP⁺ for 7 days resulted in a decrease by morethan 50% in DA uptake levels (FIG. 48). Pretreatment with 0.6 mM VPA for48 hours blocked the MPP⁺-induced degeneration of DA neurons. However,the neuroprotective effect is less pronounced than the protectionagainst LPS-induced neurotoxicity in neuron-glial cultures, whichcontaining 48% astroglia. In addition, neuron-enriched cultures treatedwith VPA alone showed much less neurotrophic effects than that found inneuron-glial cultures, again suggesting that VPA-induced neurotrophicand neuroprotective effects were dependent on the presence of astroglia.

Example 27 The HDAC Inhibitor Sodium Butyrate Mimics the NeurotrophicEffect of VPA on DA Neurons

HDAC is inhibited by therapeutically relevant concentrations of VPA andplays important roles in gene regulation; it could be the target ofVPA-induced neuronal survival-promoting effects. We then asked whetheran established HDAC inhibitor induces neurotrophic effect in midbrainneuron-glia culture similar to VPA.

Rat primary mesencephalic neuron-glia cultures seeded in a 24-wellculture plate at density of 5×10⁵ per well were treated with indicatedconcentrations of sodium butyrate or its vehicle 7 days after seeding.The viability of dopaminergic neurons was assessed by DA uptake assays 7days after sodium butyrate addition. Exposure of midbrain neuron-gliaculture to indicated concentrations of sodium butyrate had a pronouncedneurotrophic effect closely mimicked VPA in a dose-dependent manner(FIG. 49).

To explore whether astroglial cells are also the main target of sodiumbutyrate-induced neurotrophic effect, we prepared media conditioned byincubation of astroglial cultures in the absence or presence 0.6 mMsodium butyrate. Conditioned medium derived from rat primary astroglialcultures treated with vehicle (ACM) or 0.6 mM sodium butyrate(ACM-Sodium butyrate) were harvested after 48 hours of incubation.Midbrain neuron-enriched cultures seeded in 24-well plates at a densityof 5×10⁵ cells/well were treated with vehicle, sodium butyrate, ACM orACM-Sodium butyrate for 7 days. Seven days after treatment, neurotrophiceffect for dopaminergic neurons was assessed by [H]DA uptake assays. Theconditioned medium derived from incubation of astrocytes in the presenceof 0.6 mM sodium butyrate (ACM-sodium butyrate) displayed a more robustincrease in DA uptake, compared with ACM in mesencephalicneuron-enriched cultures (FIG. 50). Moreover, exposure of theneuron-enriched cultures to 0.6 mM sodium butyrate for 7 days caused aless than 2-fold increase in DA uptake, suggesting that a direct actionon neurons does not play a major role in sodium butyrate-inducedneurotrophic effects.

Quantified results are expressed as mean ±SEM of percentage ofvehicle-treated cultures from three experiments performed in duplicate.*, p<0.05 compared with the corresponding vehicle-treated controlcultures.

Example 28 3-HM is Neurotrophic to Dopaminergic Neurons

Mesencephalic neuron-glia cultures, prepared as described above, werepretreated with vehicle or 3-HM (1-5 μM) before the treatment of LPS (10ng/ml). Seven days later, the degeneration of dopaminergic neurons wasdetermined by the functional assay of [³H] DA uptake and by themorphometric measurement of dopaminergic neurons followingimmunostaining with an anti-TH antibody. [³H] DA and immunostaining areperformed as described above.

As shown in FIG. 51, the results indicated that LPS reduced DA uptakecapacity by ˜60% compared with the vehicle-treated control cultures.3-HM significantly attenuated the LPS-induced reduction in DA uptake, ina dose-dependent manner. At 5 μM, 3-HM reversed the LPS-induced decreasein DA uptake almost back to the vehicle-treated control values. Moreinterestingly, treatment with 3-HM (1-5 μM) alone for 7 daysdose-dependently increased DA uptake capacity by 20-60% compared withthe vehicle-treated control cultures, indicating that 3-HM exerted aneurotrophic effect on dopaminergic neurons.

Results from the morphometric measurements revealed a pattern of changessimilar to that of the DA uptake studies. Cell count analysis showedthat LPS reduced the number of dopaminergic neurons by 51% compared withthe vehicle-treated control cultures (FIG. 52). Pretreatment with 3-HM(5 μM) significantly restored the LPS-induced reduction in the number ofdopaminergic neurons to 97% of the vehicle-treated control cultures(FIG. 52). The average length of dopaminergic neuronal neurites in theLPS-treated cultures was 43% of the vehicle-treated control cultures,and 3-HM pretreatment increased the length to 110% of thevehicle-treated control cultures (FIG. 52). As shown in FIG. 53,following the LPS treatment, in addition to the reduction in theabundance of dopaminergic neurons, the neurites of the remaining TH-irneurons became shorter, lighter-stained, or even fragmented. Followingpretreatment with 3-HM (5 μM), dopaminergic neurons were significantlymore numerous and their neurites were less affected compared with theLPS-treated cultures. These morphological findings were consistent withthe results of the functional assay of DA uptake mentioned above.

Example 29 Neurotrophic Effect of 3-HM is Glia-Dependent and Astroglia,not Microglia, Contribute to the Neurotrophic Effect of 3-HM

One of the most interesting findings of this study was that 3-HM aloneexerted a significant neurotrophic effect in the mesencephalicneuron-glia cultures. This effect was not observed with its parentcompound DM. To determine the target of 3-HM's neurotrophic effect, wefirst investigated whether 3-HM has a direct effect on dopaminergicneurons using neuron-enriched cultures.

Various concentrations of 3-HM (0.1-5 μM) or vehicle were added to thefollowing different cell cultures: neuron-enriched cultures (A);reconstituted cultures by adding 10% and 20% (5×10⁴/well and 1×10⁵/well)of microglia to the neuron-enriched cultures (B); reconstituted culturesby adding 40% and 50% (2×10⁵/Well and 2.5×10⁵/well) of astroglia to theneuron-enriched cultures (C). Cultures are prepared as described inExample 25. The [³H]DA uptake measurements were performed 10 days aftertreatment. Results were expressed as a percentage of the vehicle-treatedcontrol cultures and were the mean ±SE from five (A) and four (B,C)independent experiments in triplicate. *P<0.05 and **P,0.001 comparedwith the vehicle-treated control cultures. #P<0.05 compared with theaugmented cultures with 40% astroglia. N, neuron-enriched cultures;N+10% (20%) MG: 10% (20%) of microglia were added back to theneuron-enriched cultures; N+40% (50%) AS: 40% (50%) of astroglia wereadded back to the neuron-enriched cultures.

3-HM (0.1-5 μM) failed to show a significant increase in the DA uptakecapacity, indicating that the observed 3-HM-induced neurotrophic effectwas not due to a direct effect on dopaminergic neurons (FIG. 54).

To examine the possibility that glia cells mediated the 3-HM-inducedneurotrophic effect, we performed reconstitution experiments by addingeither microglia or astroglia back to the neuron-enriched cultures.Addition of 10% (5×10⁴/well) or 20% (1×10⁵/well) of microglia back tothe neuron-enriched cultures failed to increase DA uptake in the31-HM-treated cultures (FIG. 55). (Normal mesencephalic neuron-gliacultures contain ˜10% microglia). In contrast, addition of 40%(2×10⁵/well) or 50% (2.5×10⁵/well) of astroglia back to theneuron-enriched cultures increased the capacity of DA uptake by 135.8%and 158.3%, respectively. (Normal mesencephalic neuron-glia culturescontain ˜40-50% astroglia). Furthermore, it appeared that theneurotrophic effect of 3-HM was positively correlated with thecomposition of astroglia (FIG. 56).

Example 30 3-HM-Treated Astroglia Conditioned Media Increase DA UptakeCapacity in the Neuron-Enriched Cultures

To confirm the possible role of astroglia in the neurotrophic effect of3-HM, conditioned media from astroglia-enriched cultures (prepared asdescribed above) treated with either 3-HM (1-5 μM) or vehicle for 24hours were prepared. These 3-HM and the vehicle-treated conditionedmedia were then added to the neuron-enriched cultures in which a newvehicle-treated control culture was viewed as the non-conditionedcontrol. Ten days later, we conducted a DA uptake assay. As shown inFIG. 57, 3-HM (1-5 μM)-treated conditioned media exerted a significantneurotrophic effect on dopaminergic neurons (151.3%, 160.9% and 197.8%,respectively) compared with the non-conditioned control cultures, in adose-dependent manner. 3-HM (2.5-5 μM)-treated conditioned media had adramatic neurotrophic effect on dopaminergic neurons (160.9% and 197.8%,respectively) compared with the vehicle-treated conditioned controlcultures (122%). However, DM (5 μM), the parent compound of 3-HM, failedto exhibit any neurotrophic effect compared with the vehicle-treatedconditioned cultures (FIG. 57). This result is consistent with ourprevious report indicating that DM by itself has no neurotrophic effect.

Results were expressed as a percentage of the vehicle-treatednon-conditioned control cultures and were the mean ±SEM from fourindependent experiments in triplicate. *P<0.05 and **P<0.001 comparedwith the vehicle-treated non-conditioned control cultures. cm,conditioned medium; non-cm, non-conditioned medium.

Treating the mesencephalic neuron-glia cultures with a relatively highdose at 1-5 μM, 3-HM has a neurotrophic effect on dopaminergic neuronsagainst LPS-induced neurotoxicity.

The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. Since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended. All references, patentapplications, patents referred to herein are hereby incorporated byreference.

1. A method of treating a mammal for endotoxic shock by administering tothe mammal in need thereof an effective amount of naloxone, wherein theeffective amount of naloxone is from 10 pg/kg to 1 μg/kg, therebytreating the endotoxic shock.
 2. The method of claim 1, wherein themammal is a human.